Method of making biologically active alpha-beta peptides

ABSTRACT

Described is a method of fabricating biologically active, unnatural polypeptides. The method includes the steps of selecting a biologically active polypeptide or biologically active fragment thereof having an amino acid sequence comprising α-amino acid residues, and fabricating a synthetic polypeptide that has an amino acid sequence that corresponds to the sequence of the biologically active polypeptide, but wherein about 14% to about 50% of the α-amino acid residues found in the biologically active polypeptide or fragment of step (a) are replaced with β-amino acid residues, and the α-amino acid residues are distributed in a repeating pattern.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of co-pending application Ser. No. 12/578,993,filed Oct. 14, 2009, which claims priority to provisional applicationSer. No. 61/106,205, filed Oct. 17, 2008, and provisional applicationSer. No. 61/229,325, filed Jul. 29, 2009, both of which are incorporatedherein by reference.

FEDERAL FUNDING STATEMENT

This invention was made with government support awarded under GM056414by the National Institutes of Health The government has certain rightsin the invention.

FIELD OF THE INVENTION

The invention is directed to a method of making polypeptide compoundscomprising alpha- and beta-amino acid residues, the compounds producedthereby, and use of the compounds as pharmaceutically active agents totreat diseases in animals, including humans.

BACKGROUND

Many naturally occurring, biologically active compounds are proteins orpeptides based upon α-amino acids (i.e., sequences of α-amino acids inwhich the α-carboxyl group of one amino acid is joined by an amide bondto the α-amino group of the adjacent amino acid). In recent years anapproach to the discovery of new pharmaceutically active drugs has beento synthesize libraries of peptides and then to assay for compoundswithin the library which have a desired activity, such as a desiredbinding activity. However, α-amino acid peptides are not altogethersatisfactory for pharmaceutical uses, in particular because they areoften poorly absorbed and subject to proteolytic degradation in vivo.

Much work on β-amino acids and peptides synthesized from β-amino acidshas been reported in the scientific and patent literature. See, forexample, the work performed by a group led by current co-inventor SamuelH. Gellman, including: “Application of Microwave Irradiation to theSynthesis of 14-helical Beta-Peptides.,” Murray & Gellman,” OrganicLetters (2005) 7(8), 1517-1520; “Synthesis of 2,2-DisubstitutedPyrrolidine-4-carboxylic Acid Derivatives and Their Incorporation intoBeta-Peptide Oligomers.,” Huck & Gellman, J. Org. Chem. (2005) 70(9),3353-62; “Effects of Conformational Stability and Geometry ofGuanidinium Display on Cell Entry by Beta-Peptides,” Potocky, Menon, &Gellman, Journal of the American Chemical Society (2005) 127(11):3686-7;“Residue requirements for helical folding in short alphabeta-peptides:crystallographic characterization of the 11-helix in an optimizedsequence,” Schmitt, Choi, Guzei, & Gellman, Journal of the AmericanChemical Society (2005), 127(38), 13130-1 and “Efficient synthesis of abeta-peptide combinatorial library with microwave irradiation,” Murray,Farooqi, Sadowsky, Scalf, Freund, Smith, Chen, & Gellman, Journal of theAmerican Chemical Society (2005), 127(38), 13271-80. Another group, ledby Dieter Seebach in Zurich, Switzerland, has also published extensivelyin the beta-polypeptide field. See, for example, Seebach et al. (1996)Helv. Chim. Acta. 79:913-941; and Seebach et al. (1996) Helv. Chim.Acta. 79:2043-2066. In the first of these two papers Seebach et al.describe the synthesis and characterization of a β-hexapeptide, namely(H-β-HVal-β-HAla-β-HLeu) 2-OH. Interestingly, this paper specificallynotes that prior art reports on the structure of β-peptides have beencontradictory and “partially controversial.” In the second paper,Seebach et al. explore the secondary structure of the above-notedβ-hexapeptide and the effects of residue variation on the secondarystructure.

Dado and Gellman (1994) J. Am. Chem. Soc. 116:1054-1062 describeintramolecular hydrogen bonding in derivatives of β-alanine and γ-aminobutyric acid. This paper postulates that β-peptides will fold in mannerssimilar to α-amino acid polymers if intramolecular hydrogen bondingbetween nearest neighbor amide groups on the polymer backbone is notfavored.

Suhara et al. (1996) Tetrahedron Lett. 37(10):1575-1578 report apolysaccharide analog of a β-peptide in which D-glycocylaminederivatives are linked to each other via a C-1β-carboxylate and a C-2α-amino group. This class of compounds has been given the trivial name“carbopeptoids.”

Regarding methods to generate combinatorial libraries, several reviewsare available. See, for instance, Ellman (1996) Acc. Chem. Res.29:132-143 and Lam et al. (1997) Chem. Rev. 97:411-448.

In the recent patent literature relating to β-polypeptides, see, forexample, U.S. published patent applications 2008/0166388, titled“Beta-Peptides with Antifungal Activity”; 2008/0058548, titled ConciseBeta2-Amino Acid Synthesis via Organocatalytic Aminomethylation”;2007/0154882, titled “Beta-polypeptides that inhibit cytomegalovirusinfection”; 2007/0123709, titled “Beta-amino acids”; and 2007/0087404,titled “Poly-beta-peptides from functionalized beta-lactam monomers andantibacterial compositions containing same.” See also U.S. publishedpatent application 2003/0212250, titled “Peptides.”

SUMMARY OF THE INVENTION

The invention is directed to a method of fabricating biologicallyactive, proteoloytic-resistant, unnatural polypeptides. The methodcomprises selecting a biologically or pharmacologically activepolypeptide or biologically active fragment thereof (the “target”)having an amino acid sequence consisting essentially of α-amino acidresidues. Then, a synthetic polypeptide is fabricated that has an aminoacid sequence that corresponds to the α-amino acid sequence of thetarget. However, in the synthetic polypeptide, between about 14% andabout 50% of the α-amino acid residues found in the target are replacedwith β-amino acid residues. More preferably between about 20% and about50% of the α-amino acid residues found in the target are replaced withβ-amino acid residues. The β-amino acid residues are disposed in thesynthetic polypeptide such that the β-amino acid residues and theα-amino acid residues are distributed in a repeating pattern throughoutthe amino acid sequence of the synthetic polypeptide. The resultingunnatural polypeptides preferably have a length of from about 10 toabout 100 residues, and more preferably of from about 20 to about 50residues. Preferably, at least two residues are β-amino acid residues.

In one version of the invention, at least one of the α-amino acidresidues in the target is replaced with at least one β-amino acidresidue that is cyclically constrained via a ring encompassing its β²and β³ carbon atoms. In another version of the invention, most or all ofthe inserted β-amino acid residues are cyclically constrained via a ringencompassing its β² and β³ carbon atoms. In another version of theinvention, at least one of the β-amino acid residues is unsubstituted atits β² and β³ carbon atoms. Alternatively all of the β-amino acidresidues may substituted at their β² and β³ carbon atoms (with linear,branched or cyclic substituents).

In another version of the invention between about 14% and about 50% ofthe α-amino acid residues found in the target are replaced with β-aminoacid residues wherein each β-amino acid residue has at least one sidechain identical to the α-amino acid residue it replaces. Thus, in thisversion, the method comprises selecting the target to be mimicked andthen fabricating a synthetic polypeptide that has an amino acid sequencethat corresponds to the sequence of the target, but wherein betweenabout 20% and about 50% of the α-amino acid residues found in the targetare replaced with analogous β-amino acid residues. In this version ofthe invention, each analogous β-amino acid residue has at least one sidechain identical to the α-amino acid residue it replaces. Again, theβ-amino acid residues and the α-amino acid residues are distributed in arepeating pattern in the amino acid sequence of the syntheticpolypeptide.

Also included within the invention are isolated, unnatural polypeptidescomprising a primary amino acid sequence as shown in SEQ. ID. NOS: 4-11,16-22, and 25-30. These unnatural polypeptides can be used in a methodof inhibiting fusion of human immunodeficiency virus to human cells. Themethod comprises contacting human cells with an isolated, unnaturalpolypeptide comprising a primary amino acid sequence as shown in SEQ.ID. NOS: 4-11, 16-22, and 25-30, whereby the cells are then resistant toentry of HIV through their cell membrane.

Another version of the invention is directed to a method of inhibitingfusion of human immunodeficiency virus (HIV) to human cells. The methodcomprises first selecting a natural, biologically active polypeptide orbiologically active fragment thereof having an amino acid sequencecomprising α-amino acid residues, and necessary for HIV fusion in vivo.A synthetic polypeptide is then fabricated that has an amino acidsequence that corresponds to the sequence of the biologically activepolypeptide or fragment thereof. In the synthetic polypeptide, betweenabout 14% and about 50% of the α-amino acid residues found in thebiologically active polypeptide or fragment are replaced with β-aminoacid residues. Further still, in the synthetic polypeptide the β-aminoacid residues and the α-amino acid residues are distributed in arepeating pattern. Human cells are then contacted with the syntheticpolypeptide.

In all embodiments of the invention, it is generally preferred (althoughnot required) that the repeating pattern places the β-amino acidresidues in alignment on one side of a helix in the unnaturalpolypeptides that adopt a helical conformation. That is, in the foldedstructure adopted by the polypeptides, the repeating pattern of α- andβ-residues disposes the β-amino acid residues in alignment along oneside of the folded molecular structure when the unnatural polypeptidesadopt a helical conformation. The repeating pattern of β-amino acidresidues and α-amino acid residues may be a pattern of from two to sevenresidues in length, such as (ααααααβ), (αααααβ), (ααααβ), (αααβ), (ααβ),(ααβαααβ), (ααβαβαβ), and (αβ). All unique patterns of α- and β-aminoacid residues of from two to seven residues in length are explicitlywithin the scope of the invention.

The method can be used to fabricate polypeptide compounds via any meansof polypeptide synthesis now known or developed in the future. Usingcurrent methods of peptide synthesis, polypeptides fabricated accordingto the present method are generally less than about 100 residues long,and more preferably from between about ten total residues and about 50total residues, more preferably still between about 20 and about 50total residues. Ranges above and below these stated ranges are withinthe scope of the invention. Many commercial services, such as Abgent(San Diego, Calif., USA) offer peptide synthesis services up to about100 residues.

The sequence of side chains along the oligomer is preferably based on aprototype α-peptide (the target) having desirable biological activityagainst a disease state. The sequence of side chains may also bemodified after translation onto the α/β-peptide backbone to optimize thedesired properties of the compounds.

Each β-residue introduced into the unnatural α/β-peptide backbone canbear side chains at one of the two backbone carbons (β³ or β²) or bothof the backbone carbons. The side chains may also be cyclicallyconstrained via a ring connecting the two backbone carbons.

Of particular note in the present invention is that substitution ofα-residues in the prototype target sequence with β-residues bearing sidechains allows modification to the backbone without disrupting thesequence of side chains along the oligomer. Cyclic β-residues rigidifythe backbone and promote helical structure.

It is preferred that β-residues be evenly spaced along the entire lengthof the sequence in order to maximize the protease resistance imparted tothe oligomer by the backbone modifications. Examples of regularlyrepeating backbone patterns include, but are not limited to, (ααααααβ),(αααααβ), (ααααβ), (αααβ), (ααβ), (ααβαααβ), (ααβαβαβ), and (αβ).

Desirable properties in the final compounds include the ability tomodulate a protein-protein interaction involved in the genesis orprogression of a disease state in general and HIV entry into human cellsin particular, and improved pharmacokinetic and pharmacodynamicproperties relative to the target α-peptide sequence (e.g., better invivo half-life, biodistribution, etc.). Many of the final compoundsadopt a helical structure in solution, although a helical structure isnot required.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, 5, 6,from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice-versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods, compounds, and compositions of the present invention cancomprise, consist of, or consist essentially of the essential elementsand limitations of the invention as described herein, as well as anyadditional or optional ingredients, components, or limitations describedherein or otherwise useful in synthetic organic chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts sequences of α-peptides 1-3 and α/β-peptides 4-11. Boldresidues indicate β³-residues corresponding to their α-amino acidcounterparts; bold, underline residues are the cyclically constrainedβ-amino acid residues ACPC (X) and APC (Y). FIG. 1B depicts structuresof an α-amino acid, the corresponding β³-amino acid analog, and cyclicβ-residues ACPC (X) and APC (Z).

FIG. 2A depicts the gp41-5 protein, composed of three NHR segments andtwo CHR segments. FIG. 2B depicts the fluorescent CHR peptide used as atracer in competition FP assays (Flu=5-carboxyfluorescein). FIG. 2C is aschematic of the interaction between the Flu-CHR peptide and the 5-helixbundle formed by gp41-5. FIGS. 3A, 3B, 3C, and 3D depict circulardichroism (CD) spectra of complexes formed between NHR peptide 1 and CHRanalogs 3 (FIG. 3A), 4 (FIG. 3B), 5 (FIG. 3C), and 10 (FIG. 3D). Solidlines are spectra observed for a 1:1 mixture of the indicated oligomersat a total concentration of 20 μM in PBS at 25° C. Dashed lines are thespectra calculated for 1:1 non-interacting mixtures from CD of theindividual components.

FIGS. 4A and 4B are a comparison of the six-helix bundles observed inthe crystal structures of the newly characterized complex betweenα-peptides 1 and 3 (FIG. 4A) and the previously characterized complexbetween α-peptides 1 and 2 (FIG. 4B) (Chan, Fass, Berger, and Kim, Cell1997, 89, 263-273). The RMSD of C_(u) atoms between the two structuresis 0.7 Å. FIG. 4C depicts the crystal structure of the 1+3 complex. FIG.4D depicts the crystal structure of the 1+10 complex solved to 2.8 Åresolution. FIG. 4E depicts the crystal structure of the 1+8 complexsolved to 2.8 Å resolution. FIGS. 4F and 4G depict overlays of theall-α-peptide helix bundle-formed 1+3 with that formed by 1+10 (FIG. 4F)and 1+8 (FIG. 4G).

FIGS. 5A, 5B, and 5C depict circular dichroism (CD) spectra. FIG. 5Adepicts superimposed CD data for NHR peptide 1 and CHR peptides 3, 4, 5,8 and 10 at 20 μM concentration in PBS at 25° C. FIG. 5B depicts CDspectra of the indicated 1:1 mixtures at a total concentration of 20 μMin PBS at 25° C. (solid lines) along with the spectra calculated for 1:1non-interacting mixtures from CD measurements on the individualcomponents (dashed lines). FIG. 5C depicts temperature-dependent molarellipticity at 222 nm for the indicated complexes at 20 μM concentrationin PBS.

FIG. 6 is a graph depicting temperature dependent molar ellipticity at222 nm for 1:1 mixtures of 1+3, 1+4, 1+5 and 1+10 at 20 μM total peptidein PBS.

FIG. 7A depicts the primary sequence of the Puma BH3 peptide (1′) andα/β-peptide analogs 2′-8′ (gray circles and bold letters indicate β³residues). FIG. 7B depicts a helical wheel diagram of 1′. Boxed residuesin FIGS. 7A and 7B indicate hydrophobic positions most important forbinding based on sequence homology. FIG. 7C presents schematicrepresentations of 1′-8′, drawn in the same orientation as in FIG. 7B;white and gray circles indicate heptad positions occupied by α-residuesand β³-residues, respectively. FIG. 7D presents the structures of ageneric α-amino acid and a generic β³-amino acid; the “R” substituent isconventionally referred to as the “side-chain.”

FIG. 8 is a histogram depicting inhibition constants for displacement ofa fluorescently labeled Bak BH3 peptide bound to Bcl-x_(L) or Mcl-1 bycompounds 1′-8′. Broken bars indicate compounds binding tighter thandiscernable in the assay. The values for 8′ were weaker than 100 μM forboth proteins.

FIGS. 9A, 9B, and 9C depict proteolytic stability of 3, 4 and 10,respectively, whose structures are shown in FIG. 9D. Solutions of 20 μMpeptide in TBS were incubated at room temperature with 10 μg/mLproteinase K. FIGS. 9A, 9B, and 9C depict time-dependent degradationdata with curves resulting from fits to a simple exponential decay. FIG.9D shows the structure of compounds 3, 4, and 10 and also depictsproteolysis products observed by mass spectrometry at the indicated timepoint. Vertical lines indicate observation by MALDI-MS of one or bothproducts consistent with hydrolysis of the backbone amide bond betweenthe indicated residues.

FIGS. 10A, 10B, 10C, and 10D are graphs depicting inhibition ofinfection of TZM-bl cells by the indicated virus strains as a functionof the concentration of gp41-derived fusion-blocking peptides. Each datapoint is the mean±S.E.M. from three independent experiments. FIG. 10Adepicts inhibition of NL4-3 infection. FIG. 10B depicts inhibition ofCC185 infection. FIG. 10C depicts inhibition of HC4 infection. FIG. 10Ddepicts inhibition of DJ258 infection.

DETAILED DESCRIPTION

The following abbreviations are used throughout the specification:

Ac₂O=acetic anhydride, acetic oxide, acetylacetate.ACPC=trans-2-aminocyclopentanecarboxylic acid.APC=trans-3-aminopyrrolidine-4-carboxylic acid.Boc=tert-butoxycarbonyl.BOP=benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate.β-Gal=β-galactosidase.CD=circular dichroism.CHR=C-terminal heptad repeat.

DIEA=N,N-diisopropylethylamine.

DMF=dimethylformamide.DMSO=dimethylsulfoxide.EDTA=ethylenediaminetetraacetic acid.FKBP=FK506-binding protein.Fmoc=9-fluorenylmethyl formyl.FP=fluorescence poloarization.

Halogen=F, Cl, Br and I.

HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluoro-phosphate.HIV=human immunodeficiency virus.

HOBT=N-hydroxybenzotriazole.

HPLC=high-performance liquid chromatography.iPr₂EtN=N,N-diisopropylethylamine.IPTG=isopropyl β-D-1-thiogalactopyranoside.MALDI-TOF-MS=matrix-assisted, laser-desorption, time-of-flight massspectrometry.MeOH=methanol.NHR=N-terminal heptad repeat.NMP=1-Methyl-2-pyrollidinone.PTH1R and PTH2R=parathyroid hormone receptors 1 and 2.RMSD=root mean square deviation.RTKs=receptor tyrosine kinases.TNF=tumor necrosis factor.PBS=phosphate-buffered saline.TBS=tris-buffered saline.Tris=tris(hydroxymethyl)aminomethane.TFA=trifluoroacetic acid.TNBS=2,4,6-trinitrobenzene-sulfonic acid.

In the present description unless otherwise indicated terms such as“compounds of the invention” embrace the compounds in salt form as wellas in free base form and also when the compounds are attached to a solidphase. Where a basic substituent such as an amine substituent ispresent, the salt form may be an acid addition salt, for example adihydrochloride. Salts include, without limitation, those derived frommineral acids and organic acids, explicitly including hydrohalides,e.g., hydrochlorides and hydrobromides, sulfates, phosphates, nitrates,sulfamates, acetates, citrates, lactates, tartrates, malonates,oxalates, salicylates, propionates, succinates, fumarates, maleates,methylene bis-b-hydroxynaphthoates, gentisates, isethionates,di-p-toluoyltartrates, methane sulfonates, ethanesulfonates,benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates,and the like. Base addition salts include those derived from alkali oralkaline earth metal bases or conventional organic bases, such astriethylamine, pyridine, piperidine, morpholine, N methylmorpholine, andthe like. Other suitable salts are found in, for example, Handbook ofPharmaceutical Salts, P. H. Stahl and C. G. Wermuch, Eds., © 2002,Verlag Helvitica Chemica Acta (Zurich, Switzerland) and S. M. Berge, etal., “Pharmaceutical Salts,” J. Pharm. Sci., 66: p. 1-19 (January 1977),both of which are incorporated herein by reference.

The β-amino acid residues of the β-peptides of the invention arecharacteristically β-amino-n-propionic acid derivatives, typicallyfurther substituted at the 2-position carbon atom (the (β² carbon)and/or the 3-position carbon atom (the β³ carbon) in the backbone andmay be further substituted, e.g., at the N-terminal amino nitrogen atom.The β², β³, and amino substituents may include substituents containingfrom 1 to 43 carbon atoms optionally interrupted by up to 4 heteroatoms, selected from O, N or S, optionally containing a carbonyl (i.e.,—C(O)—) group, and optionally further substituted by up to 6substituents selected from halo, NO₂, —OH, C₁₋₄ alkyl, —SH, —SO₃, —NH₂,C₁₋₄-acyl, C₁₋₄-acyloxy, C₁₋₄-alkylamino, C₁₋₄-dialkylamino,trihalomethyl, —CN, C₁₋₄-alkylthio, C₁₋₄-alkylsulfinyl, orC₁₋₄-alkylsulfonyl.

Substituents on the β² and/or β³ carbon atoms of β-amino acid residuesmay be selected from the group comprising the substituents which arepresent on the α-carbon atoms of natural α-amino acids, e.g., —H, —CH³,—CH(CH₃)₂, —CH₂—CH(CH₃)₂, —CH(CH₃)CH₂CH₃, —CH₂-phenyl, —CH₂-pOH-phenyl,—CH₂-indole, —CH₂—SH, —CH₂—CH₂—S—CH₃, —CH₂OH, —CHOH—CH₃,—CH₂—CH₂—CH₂—CH₂—NH₂, —CH₂—CH₂—CH₂—NH—C(NH)NH₂, —CH₂-imidazole,—CH—COOH, —CH₂—CH₂—COOH, —CH₂—CONH₂, —CH₂—CH₂CONH₂ or together with anadjacent NH group defines a pyrrolidine ring, as is found in theproteinogenic α-amino acid proline.

In accordance with the present invention it has been found that thecompounds of the invention have desirable properties. For example,compounds described herein having approximately seven or more residues,three or more of which are cyclically constrained, are able to formstable helix structures in solution. Also the compounds described hereinhave much greater stability to the action of peptidases, such as pepsin,than do their corresponding α-peptides. As such the compounds describedherein are expected exhibit correspondingly longer half lives, e.g.,serum half lives, in vivo than corresponding α-peptides.

The invention includes the compounds of the invention in pure isomericform, e.g., consisting of at least 90%, preferably at least 95% of asingle isomeric form, as well as mixtures of these forms. The compoundsof the invention may also be in the form of individual enantiomers ormay be in the form of racemates or diastereoisomeric mixtures or anyother mixture of the possible isomers.

The compounds of the invention may be prepared by the synthetic chemicalprocedures described herein, as well as other procedures similar tothose which may be used for making α-amino acid peptides. Suchprocedures include both solution and solid phase procedures, e.g., usingboth Boc and Fmoc methodologies. Thus the compounds described herein maybe prepared by successive amide bond-forming procedures in which amidebonds are formed between the β-amino group of a first β-amino acidresidue or a precursor thereof and the α-carboxyl group of a secondβ-amino acid residue or α-amino acid residue or a precursor thereof. Theamide bond-forming step may be repeated as many times, and with specificα-amino acid residues and/or β-amino acid residues and/or precursorsthereof, as required to give the desired α/β-polypeptide. Also peptidescomprising two, three, or more amino acid residues (α or β) may bejoined together to yield larger α/β-peptides. Cyclic compounds may beprepared by forming peptide bonds between the N-terminal and C-terminalends of a previously synthesized linear polypeptide.

β³-amino acids may be produced enantioselectively from correspondingα-amino acids; for instance, by Arndt-Eisert homologation of N-protectedα-amino acids. Conveniently such homologation may be followed bycoupling of the reactive diazo ketone intermediate of the Wolffrearrangement with a β-amino acid residue.

The method described herein can be used to establish discrete compoundcollections or libraries of compounds for use in screening for compoundshaving desirable activities, in particular biological activitiesindicative of particular pharmaceutical uses.

Thus the invention also includes discrete compound collections(typically comprising from 2 to about 1000 compounds) and libraries ofcompounds (typically comprising from 20 to 100 compounds up to manythousands of compounds, e.g., 100,000 compounds or more) comprisingpluralities of the compounds described herein.

Compounds having desired biological activities may be identified usingappropriate screening assays as described below.

The HIV protein gp41 is a canonical example of a class of proteinsinvolved in the fusion of enveloped viruses to mammalian cells. Duringvirus-cell fusion, the gp41 N-terminus inserts into the host cellmembrane, and the trimeric protein undergoes a drastic structuralrearrangement involving the formation a six-helix bundle composed ofthree copies of a N-terminal heptad repeat (NHR) domain and three copiesof a C-terminal heptad repeat (CHR) domain. Formation of the gp41six-helix bundle is an essential step for virus-cell fusion, and istherefore an attractive process to target for interruption using arationally designed antiviral agent. To demonstrate the utility andfunctionality of the present invention, unnatural polypeptides analogousto gp41, but comprised of mixtures of α- and β-residues (α/β-peptides)were fabricated and shown to act as inhibitors of HIV-cell fusion.

A number of α-peptides based on either gp41 NHR or CHR sequences, e.g.,compounds 1 and 2, (SEQ. ID. NOS: 1 and 2 respectively, see FIG. 1A)have been investigated as fusion inhibitors. The most prominent exampleis the 36-residue α-peptide drug enfuvirtide (sold by Hoffmann-La Roche,Inc. under the registered trademark “FUZEON”), which is derived from theCHR domain. Several groups have tried to inhibit gp41 six-helix bundleformation with short α-helix mimics, including small molecules, cyclicpeptides, terphenyls and β-peptides, that are intended to display threekey CHR hydrophobic side chains in an α-helix-like fashion; however,these molecules display only modest anti-HIV activity in cell-basedassays (IC₅₀>1 μM vs.˜1 nM for enfuvirtide). Similar results have beenseen with relatively short α-peptides that have been chemicallypredisposed toward α-helicity by internal cross-links.

The present inventors have discovered that systematically developingα/β-peptide foldamers that mimic key structural and functionalproperties of prototype α-peptide sequences, yields biologically active,unnatural polypeptides that are more stable to proteolytic degradationthan analogous α-polypeptides. The method, referred to herein as“sequence-based design,” involves the systematic substitution ofα-residues throughout a target sequence with β-amino acid residues ingeneral, and preferably β³-amino residues bearing the side chain of thereplaced α-residue. See FIG. 1B. The α→β modification alters the peptidebackbone chemical composition while retaining the side chain sequencefrom the parent α-peptide. The systematic use of sequence-based designgenerates α/β-peptides that exhibit complex behaviors such as formationof protein-like quaternary assemblies and mimicry of protein helicesinvolved in apoptosis. gp41-Mediated HIV-cell fusion was chosen as amodel system to demonstrate the utility and functionality ofsequence-based backbone modification because the target is of greatbiomedical importance. In short, a pharmacologically active agent thatinhibits gp41-mediated HIV-cell fusion, designed using a rational andsystematic method that can be repeated for other therapeuticallyimportant targets, is incredibly useful. The method provides an avenueto design pharmacologically active agents in less time, with less trialand error, and in a rational, directed fashion.

α-Peptides based on the native gp41 CHR sequence, such as compound 2,have been widely studied, and several groups have published efforts toimprove the binding affinity and biological stability of CHR α-peptidesby rational mutagenesis. To demonstrate the utility and functionality ofthe present invention, a recently reported gp41 CHR analog, α-peptide 3(SEQ. ID. NO: 3, see FIG. 1A) was chosen as the starting point for α→βmodification. α-Peptide 3 includes numerous side chain mutationsintended to enhance helical propensity by engineered intrahelical saltbridges and Xxx→Ala substitutions. In previous studies, 3 showedenhanced antiviral efficacy in cell culture and increased half-liferelative to peptides based on the wild-type CHR sequence. Although themutations in 3 were not intended to modify the structural nature of itsbinding interactions with the gp41 NHR domain, additional experimentalevidence was sought that the six-helix bundle structure was unchanged. Aco-crystal of α-peptides 1 and 3 was obtained by hanging drop vapordiffusion and the structure was solved to 2.0 Å resolution (see Table 1and FIG. 4A). The resulting six-helix bundle is essentially identical tothat formed by native NHR+CHR peptide complex. Compare FIG. 4A (the 1+3co-crystal and FIG. 4B (the 1+2 co-crystal).

TABLE 1 Crystal Data Collection and Refinement Statistics.* 1 + 3complex Data Collection Resolution (Å) 44.8-2.0 (2.1-2.0)  Totalobservations 137,233 Unique observations 15,938 Redundancy 8.6 (3.6)Completeness (%) 99.9 (100)  I/σ 28.0 (4.7)  R_(sym) ^(†) (%)  5.0(26.2) Refinement Resolution (Å) 25.0-2.0  R (%) 21.1 R_(free) ^(‡) (%)26.0 Avg. B factor (Å²) 18.6 RMSD Bonds (Å) 0.013 Angles (°) 1.1 *Valuesin parentheses are for data from the highest resolution shell;^(†)R_(sym) = Σ_(n)|I_(n) − <I>|/Σ_(n) I_(n) where I_(n) is theintensity of an independent observation of reflection n and <I> is theaverage of multiply recorded and symmetry related observations ofreflection n; ^(‡)Free R reflections (~5% of total reflections) wereheld aside throughout refinement.

Among a variety of different α/β backbone patterns examined by thepresent inventors for use in sequence-based design, the most widelystudied has been the ααβαααβ repeat. This pattern, which is tuned to theseven-residue repeat of the α-helix, places one β-residue per helicalturn and results in a “stripe” of β-residues along one face of thehelix. Crystal structures have shown that the helices formed by theααβαααβ backbone are highly homologous to the α-helix. In the initialattempt at α→β modification of 3, eleven β³-residues were incorporatedin an ccccl3 cccccc13 pattern (α/β-peptide 4, SEQ. ID. NO: 4, see FIG.1A). This resulted in the non-natural residues occupying positionsopposite the hydrophobic face involved in binding to the gp41 NHR core.

To determine the affinity of α-peptide 3 and α/β analogues for gp41, arecently reported in vitro competition fluorescence polarization (FP)assay was employed. The assay uses a protein model of the gp41 fusionintermediate. See FIGS. 2A, 2B, and 2C. The model protein, gp41-5, iscomposed of three NHR segments (SEQ. ID. NO: 12) and two CHR segments(SEQ. ID. NO: 13) linked by short flexible loops. See FIG. 2A. Thegp41-5 construct folds to form a five-helix bundle with a single bindingsite for a fluorescein-labeled CHR α-peptide. The fluorescein-labeledCHR α-peptide (SEQ. ID. NO: 14) is shown in FIG. 2B. Displacement ofthis fluorescent ligand provides the readout in competition FP. Thereaction is depicted schematically in FIG. 2C and had a displacementconstant (K_(d)) of 0.4 nM.

TABLE 2 Summary of Data Obtained for gp41 CHR Analogs 3-7, 9 and 10gp41-5 NHR + CHR Proteinase K Cell-Cell Fusion Oligomer Ki (nM)^(a) Tm(° C.)^(b) t_(1/2) (min)^(c) IC50 (nM)^(d) 3 0.2 77 0.7 9 4 3,800 e 14390 5 0.2 69 7 6 15 7 0.4 9 83 10 9 55 200 5 ^(a)Dissociation constant(K_(i)) for gp41-5 determined from competition FP experiments.^(b)Thermal unfolding transition observed by CD at 222 nm for a 1:1mixture of NHR α-peptide 1 and the indicated CHR analog in PBS at 20 μMtotal peptide concentration. ^(c)Half-life of a 20 μM solution ofpeptide in TBS in the presence of 10 μg/mL proteinase K. ^(d)IC₅₀ in acell-based fusion assay. e. No cooperative thermal transition wasobserved. --

α-Peptide 3 showed binding affinity for gp41-5 in competition FPexperiments (see Table 2) that was below the limit of detection of theassay (K_(i)<0.2 nM). α/β-Peptide analog 4 (SEQ. ID. NO: 4) showedmeasurable affinity, but it bound the model protein more than10,000-fold weaker than the prototype α sequence. Unpublished studies ofthe present inventors suggested that the W—W—I motif found near theN-terminus of 3 is critical for NHR binding. (See also Chan et al.(1998) Proc. Natl. Acad. Sci. USA 95:15613-7.) It was thus hypothesizedthat chimeric derivatives of α/β-peptide 4 which displayed these keyhydrophobic side chains on a pure α backbone (i.e., oligomers 5 and 6,SEQ. ID. NOS: 5 and 6, respectively) would show tighter binding togp41-5. Indeed, (α+α/β)-peptide 5 bound to gp41-5 with sub-nM affinityin the FP assay, indistinguishable from parent α-peptide 3. Extendingthe α/β segment in 5 toward the N-terminus (α/β-peptide 6) led to adiminution in binding affinity.

One of the fundamental motivations in the sequence-based α→βmodification of a biomedically relevant sequence such as the gp41 CHRdomain is to create oligomers that mimic the function of the parentα-peptide while displaying enhanced resistance to proteolyticdegradation. As shown herein, the ααβαααβ backbone confers useful levelsof resistance to protease; however, long α-peptide segments in chimericoligomers are effectively cleaved by proteases.

To generate α/β-peptide analogs of the gp41 CHR domain with β-residuesincorporated throughout the sequence, flexible substituted orunsubstituted β²- and/or β³-residues were replaced with cyclicallyconstrained β-residues such as trans-2-aminocyclopentanecarboxylic acid(ACPC) and trans-2-aminopyrrolidinecarboxylic acid APC, see FIG. 1B, andthose described in U.S. Pat. Nos. 6,060,585 and 6,613,876, incorporatedherein by reference. Ring constraint of the C_(α)-C_(β) bond in aβ-amino acid residue restricts torsional freedom, and promotes foldingin diverse β-peptides and α/β-peptides. Substitution of three β³-hAlaresidues in chimeric (α+α/β)-peptide 6 (SEQ. ID. NO: 6) with ACPCgenerated α/β-peptide 7 (SEQ. ID. NO: 7), which showed a 40-foldimprovement in binding to gp41-5 relative to 6. The same β³-hAla→ACPCsubstitutions were applied to oligomer 4 (SEQ. ID. NO: 4) yielding 9(SEQ. ID. NO: 9). α/β-Peptide 9 showed a 50-fold higher affinity forgp41-5 than 4. To improve binding further, three β³-hArg residues in 9were mutated to APC, a cationic analog of ACPC, to produce 10 (SEQ. ID.NO: 10). Gratifyingly, α/β-peptide 10 showed gp41-5 binding affinity(K_(d)=9 nM) that was impressive given its high degree of β-residuecontent.

α-Peptide 3 has been shown to be a potent inhibitor of HIV-cell fusionin cell culture. In the work described herein, the in vitro resultsobtained for the best α/β-peptide analog in the competition FPexperiments compared favorably to 3. The gp41 CHR mimics were thentested for their ability to block gp41-mediated membrane fusion in abiological milieu. In order to compare the efficacy of α-peptide 3 toselect foldamers in a more biologically relevant context, a previouslydescribed cell-cell fusion assay was employed. In this experiment, twocell lines are co-cultured. One cell line expresses HIV-1 Env (processedby cellular proteases to generate gp120+gp41) and Tat (an HIVtranscriptional activator). The other cell line expresses CD4 (theprimary cell surface receptor of HIV) and bears a gene for the enzymeβ-galactosidase (β-Gal) preceded by an HIV long terminal repeat sequence(sensitive to activation by Tat). Env-mediated cell-cell fusion leads toexpression of β-Gal, which can be quantified by a chemiluminescentenzymatic assay. Compounds 3, 4, 5 and 10 were tested for the ability todisrupt gp41-mediated membrane fusion in the above described assay. Theresults (Table 2) showed that the best foldamers, compounds 5 and 10(SEQ. ID. NOS: 5 and 10 respectively), have IC₅₀ values that areindistinguishable from α-peptide 3.

The interactions of select α/β-peptides with the gp41 NHR domain werefurther investigated by circular dichroism (CD) spectroscopy. The CDspectra of 3, 4, 5, 8, 9 and 10 were measured, both alone (see FIG. 5A)and in a 1:1 mixture with NHR α-peptide 1 (see FIGS. 3A, 3B, 3C, and 3D,respectively). See also FIGS. 5B and 5C for the superimposed spectra.The CHR analogs in isolation showed varying degrees of helicity.α-Peptide 3 (FIG. 3A) showed significant helical content at 20 μM inPBS, consistent with earlier published data. α/β-Peptide 10 (FIG. 3D),with seven β³>cyclic-13 substitutions, revealed an intense CD minimum,consistent with a well-folded α/β-peptide helix. The observed CDspectrum for each 1:1 mixture of NHR+CHR peptide (FIGS. 3A-3D, solidlines) was compared to that calculated by averaging spectra observed forthe corresponding individual oligomers before mixing (FIGS. 3A-3D,dashed lines). α/β-peptides 5 and 10, (SEQ. ID. NOS: 5 and 10,respectively) which showed nM or better affinity for gp41-5 in thecompetition FP assay, both showed a significant degree of inducedhelicity when mixed with NHR α-peptide 1. In contrast, α-peptide 3,which had only modest affinity for gp41-5 by FP, showed essentially nocooperative interaction with 1. The magnitude of the CD signatures amongthe well-folded mixtures (1+3, 1+5 and 1+10, FIGS. 3A, 3C, and 3D,respectively) are similar, but the ratio of intensities at 208 and 222nm changes as a function of βresidue content (more β-residues trackswith a less intense peak at 222 nm). The well-folded NHRCHR complexes(1+3, 1+5 and 1+10) each showed cooperative thermal transitions (seeFIG. 6) with Tp, values that correlate with relative differences inaffinity for gp41-5 by competition FP.

It has been shown (data omitted) that mixed α/β backbones (including theααβαααβ pattern employed in the gp41 model) can impart resistance todegradation by proteases, a serious drawback of peptide-based HIV fusioninhibitors. The stability of α-peptide 3 and α/β-peptides 4 and 10 todegradation by the promiscuous serine protease proteinase K was tested.Under the conditions of the proteolysis assay, α-peptide 3 wascompletely degraded within minutes to yield products resulting fromhydrolysis of at least ten different amide bonds in the sequence.α/β-Peptide 4, with simple α→β³ substitution, showed 20-fold improvementin stability relative to prototype α-peptide 3. α/β CHR analog 10 showedan even greater improvement in stability over α-peptide 3 (280-fold).The relative improvement in proteolytic stability of α/β-peptide 10 over4 likely results from a difference in their inherent helicity, asobserved by CD.

The present invention is thus a method employing systematic α→βmodifications to yield unnatural α/β polypeptides that retain thebiological activity of an α-amino acid prototype, yet resist proteolyticdegradation in cell culture and in vivo. As shown in the gp41 model,systematic α→β modifications in the HIV gp41 CHR domain, made inaccordance with the present invention leads to α/β-peptide analogs withpotent efficacy and enhanced proteolytic stability relative to theoriginal α-peptide. The findings establish the scope of sequence-basedbackbone modification as a general method to create oligomers that mimicthe structure and function of parent α-peptide sequences.

The gp41 model is presented herein as an illustration of how the presentinvention works in a specific environment. The method can be repeated,using any α-polypeptide as the target or prototype to be mimicked by acorresponding α/β-polypeptide fabricated according to the presentmethod.

Thus, for example, the presently claimed method can be used to fabricateα/β-polypeptides, on a rational basis, to treat rheumatoid arthritis bytargeting the interaction between tumor necrosis factor (TNF) and itsreceptor. See, for example, Williams, Ghrayeb, Feldmann, & Maini (1995)Immunology 84:433-439, for a discussion of this protein-proteininteraction. Similarly, the presently claimed method can be used tofabricate α/β-polypeptides, on a rational basis, to treat central andperipheral nervous system disorders by targeting the interaction betweengallanin and its receptor. See, for example, Mitsukawa, Lu, & Bartfai(2008) Cell. Mol. Life. Sci. (June 2008) 65(12):1796-17805 for adiscussion regarding gallanin and its receptor and the suitability ofusing this interaction to design drug targets.

The presently claimed method can also be used to fabricateα/β-polypeptides, on a rational basis, to treat disorders relating tobone and calcium metabolism by targeting the interactions betweenparathyroid hormone and its receptors (PTH1R and PTH2R). See, forexample, Usdin, Bonner, & Hoare (2002), “The parathyroid hormone 2(PTH2) receptor,” Recept. Channels 8(3-4):211-218; and Mannstadt,Juppner, & Gardella (1999), “Receptors for PTH and PTHrP: theirbiological importance and functional properties,” Am. J. Physiol. 277(5Pt. 2):F665-675.

The presently claimed method can also be used to fabricateα/β-polypeptides, on a rational basis, to treat disorders relating toserine protease reactions, such as the thrombin reaction. See, forexample, EP1141022, which describes a series of α-polypeptide thrombininhibitors. The present invention can be used to fabricateα/β-polypeptides that adopt similar conformations, have very similaranti-thrombin activity (as demonstrated in the case of the gp41 system),yet have much less susceptibility to proteolytic degradation in cellculture and in vivo.

The presently claimed method can also be used to fabricateα/β-polypeptides, on a rational basis, to inhibit the onset orprogression of neoplasms by targeting, for example, the EPH receptorsand their ephrin ligands. EPH receptors and their ephrin ligandsconstitute the largest sub-family of receptor tyrosine kinases (RTKs)and are components of cell signaling pathways involved in animaldevelopment. EPH signaling also plays an important role in oncogenicprocesses observed in several organs. These receptors are involved in awide range of processes directly related to tumorigenesis andmetastasis, including cell attachment and shape, migration, andangiogenesis. Accordingly, EPH expression and signaling activity is acritical system in the tumorigenic process. See, for example, Castano,Davalos, Schwartz & Arango (August 2008) Histol. Histopathol.23(8):1011-1023. Thus, the present method can be used to fabricateα/β-polypeptides, on a rational basis, that mimic ephrin ligands.

Once suitable drug candidates are identified, their biological and/orpharmacological activities may be assayed using any number of well-knownand industry-accepted assays.

The anti-inflammatory and immunosuppressive activities of the compoundsdescribed herein are determined by means of the following and similarassays: the IL-1β secretion inhibition, LPS fever, cytokine release fromTHP-1 cells, and functional IL-1 antagonist assays and the assay ofcarrageenan-induced paw edema in the rat (as described in EP0606044 andEP0618223); the macrophilin binding, Mixed Lymphocyte Reaction (MLR),IL-6 mediated proliferation, localized graft-versus-host (GvH) reaction,kidney allograft reaction in the rat, experimentally induced allergicencephalomyelitis (EAE) in the rat, Freund's adjuvant arthritis, FKBPbinding, steroid potentiation and Mip and Mip-like factor inhibitionassays (as described in WO9409010, EP0296123 and EP0296122).

The central nervous system (CNS) activity of the compounds describedherein is determined by means of the following and similar assays:serotonin ID (5HT 10) receptor agonist assays including the method ofWeber et al., Schmiedeberg's Arch. Pharmacol.

337, 595-601 (1988), and as described in EP0641787; 5HT 3 receptoragonist assays (as described in GB2240476 and EP0189002); assays foractivity in treatment of psychotic disorders and Parkinson's disease,such as the apomorphine-induced gnawing in the rat assay and dopaminereceptor (D1 and D2) binding assays (as described in GB20206115 B);assays for dopamine receptor antagonist activity (in relation toschizophrenia and related diseases, as described in EP0483063 andEP0544240); assays for activity in relation to senile dementia andAlzheimer's disease (as described in EP0534904); assays for activity inrelation to cerebral ischemia (as described in EP0433239), and assays inrelation to gastrointestinal motility such as the peristaltic reflex inisolated guinea pig ileum and assays of anti-serotoninergic effects(specifically at the 5-HT 4 receptors) (as described in EP0505322).

Activity of the compounds described herein in relation to bone andcalcium metabolism is determined by assays as (or similar to) thosedescribed in WO9402510, GB2218102B and WO8909786.

Activity of the compounds described herein in relation to asthma andother allergic and inflammatory conditions is determined by thefollowing assay procedures: the PDE isoenzyme inhibition, inhibition ofeosinophil activation by formyl-Met-Leu-Phe (fMLP), inhibition of TNFαsecretion, inhibition of SRS-A production, bacterial endotoxin(LPS)-induced lethality in the guinea pig, arachidonic acid-inducedirritant dermatitis in the mouse, relaxation of the human bronchus,suppression of SRS-A-induced bronchoconstriction, suppression ofbombesin-induced bronchoconstriction, suppression of methacholine(MeCH)-induced bronchoconstriction in the rhesus monkey and suppressionof airways hyperactivity in the guinea pig assays (as described in EP0664289, WO94/2493 and GB2213482).

The serine protease (e.g., thrombin) inhibition activity of thecompounds described herein is determined using assays such as thosedescribed in WO9420526. The glycoprotein IlbIIIa antagonist activity ofthe compounds described herein is determined using the assay proceduresdescribed by Cook et al., Thrombosis and Haemostasis, 70(3), 531-539(1993) and Thrombosis and Haemostasis, 70(5), 838-847 (1993), and Mülleret al. J. Biol. Chem., 268(9), 6800-6808 (1993).

Anticancer activity of the compounds described herein is determined bythe anti-tumor activity assay as described in EP0296122 or by trialprocedures, for instance as described in GB2239178. Multi-drugresistance (MDR)-reversing activity of the subject compounds isdetermined by the assays described in EP0296122.

The relevant teachings of the patent documents and other publicationsreferred to above is incorporated herein by reference. Compoundsfabricated according to the present invention which have appropriatelevels of activity in these assays are useful as pharmaceuticals inrelation to the corresponding therapies or disease states.

Thus the invention includes compounds as described herein for use aspharmaceuticals and the use of the compounds for the manufacture of amedicament for the treatment of any disease associated with any of theassays described herein, including infection by the HIV virus. Theinvention also includes the use of a compound fabricated according tothe claimed method as a pharmaceutical, and pharmaceutical compositionscomprising an effective amount of such a compound together with apharmaceutically acceptable diluent or carrier.

The compounds of the invention may be synthesized using solid phasesynthesis techniques.

Thus Fmoc-N-Protected β-amino acids can be used to synthesizepoly-α/β-peptides by conventional manual solid-phase synthesisprocedures under standard conditions on ortho-chloro-trityl chlorideresin.

Esterification of Fmoc-β-amino acids with the ortho-chloro-trityl resincan be performed according to the method of Barlos et al., TetrahedronLett. (1989), 30, 3943. The resin (150 mg, 1.05 mmol Cl) is swelled in 2ml CH₂Cl₂ for 10 min. A solution of the Fmoc-protected β-amino acid inCH₂Cl₂ and iPr₂EtN are then added successively and the suspension ismixed under argon for 4 h. Subsequently, the resin is filtered andwashed with CH₂Cl₂MeOHiPr₂EtN (17:2:1, 3×3 min), CH₂Cl₂ (3×3 min), DMF(2×3 min), CH₂Cl₂ (3×3 min), and MeOH (2×3 min). The substitution of theresin is determined on a 3 mg sample by measuring the absorbance of thedibenzofulvene adduct at 300 nm. The Fmoc group is removed using 20%piperidine in DMF (4 ml, 2×20 min) under Ar bubbling. The resin is thenfiltered and washed with DMF (6×3 min). For each coupling step, asolution of the β-amino acid (3 equiv.), BOP (3 equiv.) and HOBT (3equiv.) in DMF (2 ml) and iPr₂EtN (9 eq) are added successively to theresin and the suspension is mixed for 1 h under Ar. Monitoring of thecoupling reaction is performed with 2,4,6-trinitrobenzene-sulfonic acid(TNBS) (W. S. Hancock and J. E. Battersby, Anal. Biochem. (1976), 71,260). In the case of a positive TNBS test (indicating incompletecoupling), the suspension is allowed to react for a further 1 h. Theresin is then filtered and washed with DMF (3×3 min) prior to thefollowing Fmoc deprotection step. After the removal of the last Fmocprotecting group, the resin is washed with DMF (6×3 min), CH₂Cl₂ (3×3min), Et₂O (3×3 min) and dried under vacuum for 3 h. Finally thepeptides are cleaved from the resin using 2% TFA in CH₂Cl₂ (2 ml, 5×15min) under Ar. The solvent is removed and the oily residues aretriturated in ether to give the crude α/β-polypeptides. The compoundsare further purified by HPLC.

The oral bioavailability of the compounds described herein is determinedin the rat using standard procedures. The absolute oral bioavailabiltyis expected to be about 1%.

In view of the stable structures which α/β-peptides exhibit in solution,their stability to enzymatic degradation and their encouragingpharmacokinetic properties, the compounds of the invention have thepotential to provide useful pharmaceutical products.

As noted above, the gp41 CHR-derived α-peptide, 3 was used as thestarting point for α→β modification (FIG. 1A). α-Peptide 3, also knownas T-2635, is 50% mutated as compared to the wild type gp41 CHR domainand contains a combination of Xxx→Ala substitutions and engineered i→i+4salt bridges that were intended to enhance α-helical propensity.α-Peptide 3 represents one of the most successful examples reported todate of improving the antiviral efficacy of gp41 CHR α-peptides viamodification of the α-amino acid sequence. The initial studies beganwith the side chain sequence optimized in 3. Also explored were changesin backbone composition in the form of α→β residue substitution. Inα/β-peptide 4, a subset of the α-residues in 3 has been replaced byβ³-residues that bear the side chain of the replaced α-residue (see FIG.1B). Thus, α/β-peptide 4 has the sequence of side chains found in 3displayed on an unnatural backbone. The β³-residues of 4 areincorporated in an ααβαααβ pattern, which, upon folding, generates astripe of β-residues that runs along one side of the helix. This designplaces the β-stripe in 4 distal along the helix circumference to themolecular surface that packs against the gp41 NHR domain trimer in thesix-helix bundle.

A competition fluorescence polarization (FP) assay based on a proteinmodel of the gp41 six-helix bundle was used to compare 3 and 4. (See theExamples for details.) The assay measures displacement of afluorescently-labeled CHR α-peptide from an engineered five-helix bundleprotein, gp41-5, which contains three NHR segments and two CHR segments.Affinity for the gp41-5 protein construct correlates with the ability ofCHR-mimetic agents to bind to the gp41 pre-hairpin intermediate formedjust prior to HIV-cell fusion. As expected, α-peptide 3 binds verytightly to gp41-5 (K_(i)<0.2 nM; Table 1). The analogous α/β-peptide 4,however, displays only weak affinity for gp41-5, >10,000-fold lower thanthat of 3. The modest potency of α/β-peptide 4 in this protein-basedassay is comparable to that displayed by a number of small molecules andpeptidomimetics in comparable experiments.

In an effort to understand the dramatic differences in binding between 3and 4 and to improve the affinity of the α/β-peptide for gp41, chimericα/β-peptides 5 and 6 were prepared and characterized. Both 5 and 6contain a pure α segment at the N-terminus and an α/β segment at theC-terminus; these oligomers are chimeras of α-peptide 3 and α/β-peptide4. α/β-Peptide 5 displays very high affinity for gp41-5,indistinguishable from that of α-peptide 3; however, extending the α/βsegment toward the N-terminus (as in 6) causes a significant loss ofaffinity. The sensitivity of the N-terminal segment to sa-33modification is consistent with data showing that side chains in thisregion, especially those corresponding to Trp₃, Trp₆ and Ile₁₀ in 3,play a crucial role in CHR binding to the NHR trimer.(29)

α/β-Peptides 5 and 6 represent an improvement in gp41 mimicry relativeto 4, but it would be desirable to place β-residues throughout anα/β-peptide sequence in order to maximize resistance to proteolysis.Each α→β³ replacement, however, adds a flexible bond to the backbone,which should increase the conformational entropy penalty associated withhelix formation. The greater conformational entropy of the unfoldedstate of 4 relative to 3, arising from eleven α→β³ replacements, mayaccount for the large difference in binding affinity for gp41-5 betweenthese two oligomers. Although β-residues are the source of this loss ofstability, these residues provide an avenue for conformationalpre-organization that is made uniquely possible by their chemicalstructure. Incorporation of cyclic β-residues (e.g., ACPC and APC, FIG.1B) can constrain the C_(α)-C_(β) backbone torsion and thereby enhancefolding propensity without disrupting backbone amide hydrogen bonding.

The impact of conformational preorganization in the context of gp41mimicry was probed by replacing a subset of β³-residues with cyclicanalogues. The first comparison involved α/β-peptide 7, the analogue of6 in which the three β³-hAla residues are replaced by ACPC (FIGS. 1A and1B). Both β³-hAla and ACPC are non-polar, and this similarity wasexpected to maintain the physical properties that emerge from side chainsequence. The >30-fold higher affinity for gp41-5 displayed by 7relative to 6 supports the hypothesis that residue-based rigidificationis a useful complement to sequence-based design for developingpeptide-mimetic foldamers. Replacement of two β³-hArg residues inoligomer 7 with APC, a heterocyclic analogue of ACPC, leads toα/β-peptide 8, which showed a very high affinity for gp41-5. APC₃₆ inα/β-peptide 8 is in a region of the CHR sequence that does not engagethe NHR region contained in gp41-5; this observation may explain thesimilar K, values of 7 and 8. Additional evidence of the favorablecontribution of cyclic β-residues comes from comparison of oligomers 4,9 and 10, each of which has β-residues throughout the sequence.α/β-Peptide 9 was generated from 4 by four β³-hAla→ACPC replacements,which leads to a >45-fold improvement in K. Replacement of the threeβ³-hArg residues of 9 with APC, to generate 10, improves K, by a further˜10-fold. Relative to completely flexible α/β-peptide 4, rigidifiedanalogue 10 (K_(i)=9 nM) shows ˜380-fold enhanced binding to gp41-5.

The interactions of CHR α-peptide 3 and α/β-peptide analogues 4, 5, 8and 10 with a peptide derived from the gp41 NHR domain (1) wereinvestigated by circular dichroism (CD) spectroscopy. NHR α-peptide 1forms a six-helix bundle when mixed with gp41 CHR α-peptides; thissix-helix bundle is thought to represent the post-fusion state adoptedby gp41 in the course of viral entry. α-Peptide 3 showed significanthelical content at 20 μM in PBS, consistent with previously publisheddata (FIG. 5A). α/β-Peptide 4 showed no significant helicity undersimilar conditions; however, analogue 10, with seven β³→cyclic-βsubstitutions, showed an intense CD minimum, consistent with awell-folded α/β-peptide helix (FIG. 5A). The observed CD spectrum foreach 1:1 mixture of NHR+CHR peptide was compared (FIGS. 3A through 3D,and 5B, solid lines) to that calculated by averaging spectra for thecorresponding individual oligomers (FIGS. 3A through 3D and 5B, dashedlines). α/β-Peptides 5, 8 and 10, which displayed high affinity forgp41-5 in the competition FP assay, each showed a significant degree ofinduced helicity when mixed with NHR α-peptide 1, which is consistentwith six-helix bundle formation. By contrast, α/β-peptide 4, which hasonly modest affinity for gp41-5, showed essentially no interactionwith 1. The magnitude of the CD signatures among the well-foldedmixtures (1+3, 1+5, 1+8 and 1+10) are similar, but the ratio ofintensities at 208 and 222 nm changes as a function of β-residue content(higher (3-residue content is correlated with a less intense peak at 222nm). This trend is consistent with previous studies on helical oligomerscontaining mixed α/β backbones. The complexes formed by 1+3, 1+5, 1+8and 1+10 each showed highly cooperative thermal transitions (FIG. 5C).The trend in T_(m,app) values (i.e., apparent T_(m)) correlates withdifferences in affinity among 3, 5, 8 and 10 for gp41-5 in thecompetition FP assay; that is, stronger binding to gp41-5 correlateswith more stable assembly with NHR peptide 1.

Crystal Structures.

X-ray crystallography was employed to compare the heteromeric six-helixbundles formed by NHR α-peptide 1 with CHR α-peptide 3, chimeric CHRα/β-peptide 8 or CHR α/β-peptide 10 (see Table 3).

TABLE 3 X-ray data collection and refinement statistics 1 + 3 complex 101 + 10 complex 1 + 8 complex Data collection Space group P2₁2₁2 C2 P4₁32H32 Cell dimensions a, b, c (Å) 37.6, 179.0, 33.1 71.3, 44.0, 58.1 84.9,84.9, 84.9 57.0, 57.0, 186.3 α, β, γ (°) 90, 90, 90 90, 105.4, 90 90,90, 90 90, 90, 120 Resolution (Å) 44.8-2.0 (2.1-2.0)*  50.0-2.1(2.18-2.10)* 50.0-2.8 (2.9-2.8)* 50.0-2.8 (2.9-2.8)* R_(sym) (%)  5.0(26.2)  6.7 (35.6)  6.1 (51.2)  5.8 (38.8) I/σI 28.0 (4.7)  16.1 (3.5) 31.6 (2.8)  16.8 (3.7)  Completeness 99.9 (100)  99.8 (99.8) 99.8 (98.2)99.5 (100)  (%) Redundancy 8.6 (3.6) 3.5 (3.4) 7.8 (6.2) 5.9 (6.2)Refinement Resolution (Å) 25.0-2.0  25.0-2.1  25.0-2.8  25.0-2.8  No.reflections 15,123 9,769 2,730 2,947 R_(work) /R_(free) (%) 20.9/26.020.4/24.9 26.6/30.7 25.2/31.1 Avg. B factor (Å²) RMSD Bond lengths 0.0130.015 0.013 0.018 (Å) Bond angles (°) 1.1 2.0 1.7 1.8 *Highestresolution shell is shown in parenthesis.

Although the mutations to the native CHR sequence that lead to α-peptide3 were not intended to modify the nature of its binding interactionswith the gp41 NHR domain, direct evidence was sought that the six-helixbundle structure was unchanged relative to that formed by 1 and thenative CHR sequence. A co-crystal of α-peptides 1 and 3 was obtained andits structure solved to 2.0 Å resolution. See FIG. 4A. The resultingsix-helix bundle is essentially identical to that for 1+2 (see FIG. 4B)which contains the native CHR sequence; the root mean square deviation(rmsd) is 0.73 Å for C_(a) atoms.

A crystal of the 1+10 complex was also obtained and its structure solvedto 2.8 Å resolution. See FIG. 4D. α-Peptide 1 and α/β-peptide 10 combineto form a six-helix bundle that is similar to the assembly formed by 1+3(duplicated in FIG. 4C to allow a side-by-side comparison). A crystalcontaining only α/β-peptide 10 (not shown) was obtained as well. Thestructure of 10 alone, solved to 2.1 Å resolution, revealed a paralleltrimeric helix bundle with a hydrophobic core comprising the residuesthat engage the gp41 NHR trimer in the six-helix bundle formed by 1+10.The self-assembly of cup-peptide 10 in the crystalline state parallelsthe behavior previously observed for prototype α-peptide 3, which wasshown to self-assemble in solution.

The core NHR trimers in the structures of 1+10 and 1+3 are highlyhomologous (0.65 Å C_(α) rmsd for NHR residues 3-30). When the twobundles are aligned via the NHR trimer, the CHR helices track veryclosely in the C-terminal segment (0.84 Å C_(α) rmsd for residues 16-33)but diverge near the N-terminus (4.2 Å C_(α) rmsd for residues 2-15).This divergence reflects a greater superhelical twist in α-peptide 3relative to α/β-peptide 10. The divergent portion of the helix formed by10 contains the two Trp residues that, in CHR α-peptides, are essentialfor stable six-helix bundle formation. In the structure of 1+10, theside chains of Trp₃ and Trp₅ were not resolved in electron density,suggesting a high degree of disorder. In addition, significant disorderwas observed in the side chains of NHR residues Lys₂₉ and Trp₂₆, whichpack around CHR Trp₅ in the 1+3 complex. FIGS. 4F and 4G depict overlaysof the all-α-peptide helix bundle-formed 1+3 with that formed by 1+10(FIG. 4F) and 1+8 (FIG. 4G).

Given the well-established role of the gp41 CHR domain Trp-Trp-Ile motifin six-helix bundle formation, the observation that the N-terminalsegment of α/β-peptide 10 does not engage the NHR binding pocket in thecrystal structure of the 1+10 complex is intriguing. Removal of thefirst ten residues of α/β-peptide 10 leads to oligomer 11, in which theTrp-Trp-Ile motif is not present (see FIG. 1A). If the N-terminal regionof 10 were not involved in binding to the NHR trimer in solution, asmight be suspected based on the crystal structure of 1+10, then 11should show affinity for gp41-5 that is comparable to that of 10.However, α/β-peptide 11 showed no measurable affinity for gp41-5(K_(i)>10 μM), indicating that the N-terminal segment of 10 is essentialfor high-affinity binding to gp41-5 in solution.

Motivated by the differences between the CHR domain N-terminal segmentsin the 1+3 complex and the 1+10 complex, the structure of NHR peptide 1in complex with CHR α/β-peptide 8, a chimera of α-peptide 3 andα/β-peptide 10, was investigated. The 1+8 complex was crystallized andits structure solved to 2.8 Å resolution. See FIG. 4E. Relative toα/β-peptide 10, chimeric α/β-peptide 8 tracks much more closely with theCHR helix (3) in the all-α-peptide, six-helix bundle formed by 1+3 (FIG.4C, 1.4 Å C_(α) rmsd for residues 2-33). The side chains of theTrp-Trp-Ile motif in the N-terminal segment of 8 show the expectedpacking into the binding pocket on the NHR core trimer (data not shown).Based on this result and the behavior of truncated α/β-peptide 11, it issuspected that the lack of direct contact between the N-terminal portionof 10 and the NHR trimer in the 1+10 complex is an artifact of crystalpacking.

Antiviral Activity.

Two sets of experiments were performed to evaluate the activities ofα-peptide 3 and α/β-peptides 4, 5 and 10 in a biological context. Thefirst experiment compared the oligomers in a cell-cell fusion assaybased on expression of the env gene of the HIV-1 clone HxB2, an assaythat is commonly used to model gp41-mediated HIV-cell fusion. (Deng Y Q,Zheng Q, Ketas T J, Moore J P, & Lu M (2007) Protein design of abacterially expressed HIV-1 gp41 fusion inhibitor. Biochemistry46(14):4360-4369.) The cell-cell fusion assay results (Table 4) showedthat α/β-peptides 5 and 10 have IC₅₀ values indistinguishable from thatof α-peptide 3, while α/β-peptide 4 is much less effective. Compounds 3,4, 5 and 10 were then evaluated for the ability to prevent HIV infectionof the cell line TZM-bl. (Wei X P, et al. (2002) Emergence of resistanthuman immunodeficiency virus type 1 in patients receiving fusioninhibitor (T-20) monotherapy. Antimicrob Agents Chemother46(6):1896-1905.) These studies employed one T-cell line adapted strainand three primary isolates; two of the strains are X4-tropic, and theother two are R5-tropic.

TABLE 4 Summary of physical and functional data obtained for gp41 CHRanalogues 3-11. gp41-5 binding NHR + CHR Cell-cell affinity stabilityStability to fusion Inhibition HIV-1 infectivity, IC₅₀ (nM) ^(e) by FP^(a) by CD ^(b) Proteinase K ^(c) inhibition ^(d) X4 strains R5 strainsOligomer K_(i) (nM) T_(m, app) (° C.) t_(1/2) (min.) IC₅₀ (nM) NL4-3 HC4CC 1/85 DJ258 3 <0.2 77 0.7 9 ± 3   5 ± 0.6 27 ± 4 140 ± 20 58 ± 6 43,800 — ^(f) 14 390 ± 40  700 ± 60  590 ± 100 1300 ± 100  960 ± 200 5<0.2 67 7 ± 2 10 ± 2 55 ± 8 270 ± 20 280 ± 90 6 15 7 0.4 8 0.3 65 9 8310 9 55 200 5 ± 2 28 ± 3  59 ± 10 180 ± 30 110 ± 40 11 >10,000 T-20  700± 100 250 ± 20 1400 ± 400 330 ± 60 ^(a) Dissociation constant (K_(i))for binding to the protein gp41-5 as determined by competition FPexperiments. ^(b) Melting temperature (T_(m, app)) for the thermalunfolding transition observed by CD at 222 nm for a 1:1 mixture of NHRα-peptide 1 and the indicated CHR analogue at 20 μM total peptideconcentration in PBS. ^(c) Half-life (t_(1/2)) of a 20 μM solution ofpeptide in TBS in the presence of 10 μg/mL proteinase K. ^(d) Values arethe means ± S.E.M. of IC₅₀ values obtained in three independentexperiments. The envelope protein expressed was of the HxB2 clone,derived from the T-cell-line-adapted isolate IIIB of clade B. ^(e)Values are the means ± S.E.M. of IC₅₀ values obtained in threeindependent experiments. ^(f) The temperature dependent CD for the 1 + 4mixture was not significantly different than that calculated from theaverage of the temperature dependent CD spectra of 1 alone and 4 alone.The results of the infectivity assays (Table 4, FIGS. 10A, 10B, 10C, and10D) show similar biological potencies among 3, 5 and 10 for HIV-1strains that use different co-receptors. This finding indicates theblocking of a necessary, shared step in entry through peptideinteractions with conserved regions of gp41. It may be noted that thereis imperfect correlation between K_(i) for binding to gp41-5 and IC₅₀values in cell-based assays among the compounds reported here. Forexample, the affinity of 10 for gp41-5 was >45-fold higher than that of5, yet IC₅₀ values for 10 were sometimes lower than for 5. There areseveral possible reasons for this discrepancy. Sequence differencesbetween the CHR and NHR domains found in gp41-5 and those found in theviruses tested may lead to better correlation between gp41-5 bindingaffinity and antiviral activity against some strains relative to others.In addition, it has previously been suggested that the association ratesfor CHR peptides binding to gp41 are a better predictor of relativeantiviral potencies than are equilibrium binding affinities. (Steger H K& Root M J (2006) Kinetic dependence to HIV-1 entry inhibition. JBiolChem 281(35):25813-25821.) The rigidified backbone in 10 may alter itsassociation rate with gp41 relative to that of 5. Sensitivity togp41-derived fusion inhibitors may be affected by many factors thatdiffer among strains of virus, including the amount of Env incorporatedinto the virion, the strength of Env interactions with CD4 and withco-receptors, the kinetics and energetics of the fusion process, as wellas amino acid variation in the binding site for inhibitory peptides.Overall, the antiviral assays results strongly support the hypothesisthat CHR-derived α/β-peptides effectively mimic gp41 in a complexbiological milieu.

Proteolytic Susceptibility.

An important motivation for developing foldamer antagonists ofprotein-protein interactions is the prospect of diminishing sensitivityto proteolytic degradation. Rapid destruction by proteolytic enzymesrepresents a significant drawback to the clinical use of α-peptidedrugs. The susceptibilities of α-peptide 3 and α/β-peptides 4 and 10 todegradation by proteinase K, a promiscuous serine protease, werecompared. Under the assay conditions, α-peptide 3 was completelydegraded within minutes (FIG. 9A); mass spectrometry revealed hydrolysisof at least ten different amide bonds in the sequence (FIG. 9D, topsequence). α/β-Peptide 4, with exclusively α→β³ substitution, showed20-fold improvement in stability relative to prototype α-peptide 3. SeeFIG. 9B and FIG. 9D, middle sequence. Rigidified α/β-peptide 10 showedan even greater improvement in stability over α-peptide 3 (280-fold).See FIG. 9C and FIG. 9D, bottom sequence. The greater stability ofα/β-peptide 10 relative to α/β-peptide 4 likely results from the greaterhelical propensity of 10, as detected by CD. The small number ofproteolysis products observed for α/β-peptide 10 by mass spectrometry(FIG. 9D) supports previous observations that β-residues in mixed α/βbackbones tend to protect neighboring amides from proteolytic cleavage.

Many proteins display surfaces that participate in highly selectiveinteractions. Information flow mediated by protein-protein interactionsis essential for normal function of individual cells and entireorganisms; such interactions can play key roles in disease as well.There is considerable motivation to identify strategies for inhibitingthe formation of specific inter-protein complexes. At the clinicallevel, the most successful approach to this goal involves the use ofengineered proteins or protein fragments, i.e., molecules constructedfrom the same building blocks as the protein targets themselves. Themotivating hypothesis of the presently claimed method is thatrecognition surfaces displayed by proteins can be mimicked withunnatural oligomers that adopt protein-like conformations and displayprotein-like side chains, and that such oligomers will function asinhibitors of natural protein-protein associations. Natural proteinsequences are logical starting points for designing folded oligomerswith normatural backbones that have sophisticated functions. The datapresented here provide strong support for these hypotheses in thecontext of a widely studied viral infection process.

The results presented herein indicate that a long α-helical segment, theCHR region of HIV protein gp41, can be structurally and functionallymimicked by oligomers composed of α- and β-amino acid residues. Atwo-stage process was required to generate an α/β-peptide that manifestsa favorable profile of properties, including strong association with theintended binding partner, potent inhibition of HIV infection in acell-based assay and resistance to proteolytic cleavage. The firstdesign stage involves replacement of selected α-residues in a parentpeptide sequence with homologous β-residues that retain the originalside chains. The second design stage involves selective replacement offlexible β³-residues with cyclically preorganized β-residues. Thesemodifications are intended to remove deleterious backbone flexibilitythat is unavoidably introduced with the initial α→β³ modifications.

Using a two-stage approach for creation of an effective α/β-peptidemimic of the gp41 CHR segment is noteworthy in light of our previousfindings in a different and inherently simpler protein recognitionsystem. Mimicry of BH3 domains, short α-helical segments that mediateprotein-protein interactions in the Bcl-2 protein family, required onlythe first stage of this design approach, simple α→β³ substitutionthroughout the prototype sequence. (Home W S, Boersma M D, Windsor M A,& Gellman S H (2008) Sequence-based design of α/β-peptide foldamers thatmimic BH3 domains. Angew Chem Int Ed 47(15):2853-2856.) In contrast,α/β-peptide 4, which showed only modest affinity for gp41-5, was themost potent gp41 mimic identified among a series of α/β-peptidesdesigned by exploring alternative α/β³ backbone patterns in the nativegp41 CHR domain and related sequences.

The results reported here represent a substantial advance relative toearlier efforts to develop unnatural oligomers that mimic α-helicesinvolved in protein-protein recognition events. Previous work has beenlimited to relatively short α-helical targets, typically only two tofour helical turns. Efficacies of oligomers developed in these priorstudies have generally been modest (IC₅₀ values greater than 1 μM).Moreover, in most previously studied systems, effective inhibition hasbeen possible with small molecule antagonists. The present results aredistinctive because the data show that a long α-helix (˜10 turns) can bestructurally and functionally mimicked with a rationally designedoligomer. To date, efforts to disrupt gp41 six-helix bundle assemblywith small molecules have been relatively unsuccessful.

The present work demonstrates the value of designing unnatural oligomersthat can “read” the sophisticated recognition signals that have beenevolutionarily encoded in natural proteins. Potent inhibition of HIVinfectivity by α/β-peptides is an important advance in the developmentof functional foldamers.

EXAMPLES Reagents

Protected α-amino acids and resins used in peptide synthesis werepurchased from Novabiochem (a wholly owned subsidiary of EMD ChemicalsInc. and Merck KGaA, Darmstadt, Germany). Protected β³-amino acids werepurchased from PepTech (Burlington, Mass., USA). Cyclically constrainedβ-residues, Fmoc-ACPC and Fmoc-APC(Boc), were prepared as previouslydescribed. Lee, LePlae, Porter, and Gellman, J. Org. Chem. 2001, 66,3597-3599; LePlae, Umezawa, Lee, and Gellman, J. Org. Chem. 2001, 66,5629-5632. 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluoro-phosphate (HBTU) was purchased from AnaSpec (San Jose,Calif., USA). 5-Carboxyfluorescein was purchased from Invitrogen(Carlsbad, Calif., USA). 1-Methyl-2-pyrollidinone (NMP) was purchasedfrom Advanced Chemtech (Louisville, Ky., USA). All other reagents werepurchased from Sigma-Aldrich Corp. (St. Louis, Mo., USA) or FisherScientific (Pittsburgh, Pa., USA) and used as received.

Synthesis:

All peptides were prepared on “NovaSyn TGR”-brand resin (Novabiochem).α-Peptides were prepared by standard Fmoc solid phase peptide synthesismethods on a Symphony Multiple Peptide Synthesizer (ProteinTechnologies, Inc., Tucson, Ariz., USA). α/β-Peptides were prepared byautomated Fmoc solid phase peptide synthesis on a Synergy 432A automatedsynthesizer (Applied Biosystems, Foster City, Calif., USA). α/β-Peptideswere also prepared manually by microwave-assisted Fmoc solid phasepeptide synthesis. Erdelyi and Gogoll (2002) Synthesis 11:1592-1596. TheN-terminus of each peptide was capped by treatment with 8:2:1DMFDIEAAc₂O. The resin was washed thoroughly (3×DMF, 3×CH₂Cl₂, 3×MeOH)and then dried under vacuum. All peptides were cleaved from resin bytreatment with 94:2.5:2.5:1 TFAH₂O/ethanedithiol/triisopropylsilane. Theresin was filtered, washed with additional TFA, and the combinedfiltrates concentrated to 2 mL under a stream of dry nitrogen. Crudepeptide was precipitated from the cleavage mixture by addition of coldether (45 mL). The mixture was centrifuged, decanted, and the remainingsolid dried under a stream of nitrogen. Peptides were purified byreverse phase HPLC on a prep-C₁₈ column using gradients between 0.1% TFAin water and 0.1% TFA in acetonitrile. The identity and purity of thefinal products were confirmed by MALDI-TOF-MS and analytical HPLC,respectively. Stock solution concentrations were determined by UVabsorbance. Gill, S. C.; Vonhippel, P. H. Anal. Biochem. 1989, 182,319-326. MALDI-TOF-MS (monoisotopic [M+H]⁺, m/z): 1: obsd.=4162.6,calc.=4162.4; 2: obsd.=4288.7, calc.=4288.0; 3: obsd.=4455.0,calc.=4455.3; 4: obsd.=4609.9, calc.=4609.5; 5: obsd.=4526.1,calc.=4525.4; 6: obsd.=4552.7, calc.=4553.4; 7: obsd.=4631.6,calc.=4631.4; 8: obsd.=4516.5, calc.=4515.3; 9: obsd.=4713.0,calc.=4713.5; 10: obsd.=4539.9, calc.=4539.4; 11: obsd.=3299.4,calc.=3299.8.

Synthesis of Flu-C38:

“NovaSyn TGR”-brand resin bearing the full-length C38 peptide with freeN-terminus (WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDK; SEQ. ID. NO: 23) wasprepared on a 25 pmol scale by standard Fmoc solid phase peptidesynthesis methods on a Symphony Multiple Peptide Synthesizer (ProteinTechnologies, Inc.). Following synthesis, the resin was transferred to afritted syringe. 5-Carboxyfluorescein (28 mg, 0.075 mmol) and HOBTH₂O(11 mg, 0.075 mmol) were dissolved in N-methyl-2-pyrrolidinone (0.75mL). Diisopropylcarbodiimide (12 μL, 0.075 mmol) was added. Theresulting solution was transferred to the peptide-bearing resin. Thereaction vessel was covered in foil and placed on a shaker overnight.The resin was washed with DMF (3×), and the coupling reaction wasrepeated with fresh reagents. The resin was then washed with DMF (3×),20% piperidine (2×), DMF (3×), CH₂Cl₂ (3×), and MeOH (3×). Fischer, R.;Mader, O.; Jung, G.; Brock, R. Bioconjugate Chem. 2003, 14, 653-660. Thecrude peptide was cleaved and purified as described above. Stocksolutions, prepared in water, were quantified by visible absorbance(ε₄₉₄=68,000 M⁻¹ cm⁻¹ at pH 8). MALDI-TOF-MS (monoisotopic [M+H]⁺, m/z):obsd.=5089.3, calc.=5089.3.

Crystallization.

Hanging drops were prepared by mixing 1 μL of crystallization stock and1 μL of reservoir buffer followed by room temperature equilibration over0.7 mL buffer. Stock solutions of the 1+3 and 1+8 complexes wereprepared by mixing concentrated stocks of the individual peptides in a1:1 ratio to a final concentration of 2.2 mM total peptide in water.Crystals of 1+3 were obtained from a reservoir buffer comprising 0.1 MTris pH 8.5, 1 M (NH₄)H₂PO₄. Crystals of the 1+8 complex were grown areservoir buffer comprising 0.4 M Li₂SO₄H₂O, 12% vv PEG 8000, 20% vvglycerol. In initial attempts to crystallize the 1+10 complex, a stocksolution was prepared by mixing concentrated stocks of the individualpeptides in a 1:1 ratio to a final concentration of 0.76 mM totalpeptide in water. Stocks of 1+10 prepared in this way were not fullysoluble. However, the resulting viscous suspension yielded crystals ofcc13-peptide 10 alone from a well buffer comprised of 0.5 M ammoniumsulfate, 0.1 M HEPES-Na, pH 7.5, 30% vv 2-methyl-2,4-pentanediol. Forsubsequent crystallization trials of 1+10, the stock solution of thecomplex was prepared by refolding the 1:1 peptide mixture at 130 μMtotal peptide in water followed by concentration to 1.1 mM bycentrifugation at 4° C. through a 10 kDa molecular weight cutoffmembrane. Crystals of 1+10 were obtained from a stock prepared in thisway and a reservoir buffer comprised of 0.2 M NaCl, 0.1 M Tris pH 8.5,25% wv PEG 3350.

X-Ray Data Collection, and Structure Determination.

All crystals were flash frozen in liquid nitrogen. Crystals of the 1+3complex were briefly soaked in 0.08 M Tris pH 8.5, 1.6 M (NH₄)H₂PO₄, 20%vv glycerol prior to freezing. Crystals of 10 and 1+8 were frozendirectly from the crystallization drop. Crystals of the 1+10 complexwere soaked briefly in 0.2 M NaCl, 0.1 M Tris pH 8.5, 25% wv PEG 3350,20% vv glycerol prior to freezing. Diffraction data for the 1+3 and 1+8complexes were collected on a Bruker X8 Proteum Diffractometer (BrukerAXS, Inc. Madison, Wis. USA) using Cu K_(α) radiation and were processedwith the Bruker Proteum2 software package. Diffraction data for thecrystals of 10 and the 1+10 complex were collected at the Life SciencesCollaborative Access Team beamline 21-ID-G at the Advanced PhotonSource, Argonne National Laboratory, and were processed withHKL-2000-brand software (HKL Research, Inc., Charlottesville, Virginia,USA). Structure determination was carried out using the CCP4 softwaresuite. Collaborative Computational Project Number 4 (1994) The CCP4Suite—Programs for Protein Crystallography. Acta Crystallogr, Sect D50:760-763. Molecular replacement was carried out with Phaser software(McCoy A J, Grosse-Kunstleve R W, Storoni L C, & Read R J (2005)Likelihood-enhanced fast translation functions. Acta Crystallogr, Sect D61:458-464) or Molrep software (Vagin A & Teplyakov A (1997) MOLREP: Anautomated program for molecular replacement. J Appl Crystallogr30(6):1022-1025). Refinement was accomplished by a combination of Refmac(Murshudov G N, Vagin A A, & Dodson E J (1997) Refinement ofmacromolecular structures by the maximum-likelihood method. ActaCrystallogr, Sect D 53:240-255) for automated refinement, Coot (Emsley P& Cowtan K (2004) Coot: Model-building tools for molecular graphics.Acta Crystallogr, Sect D 60:2126-2132) for manual model building, andARPwARP for automated water building and free atom density modification.(Lamzin V S & Wilson K S (1993) Automated refinement of protein models.Acta Crystallogr, Sect D 49:129-147.) The structure of the 1+3 complexwas solved using a search model derived from a published gp41 hexamerstructure (PDB ID: 1AIK). Chan D C, Fass D, Berger J M, & Kim P S (1997)Core structure of gp41 from the HIV envelope glycoprotein. Cell89(2):263-273. The structure of α/β-peptide 10 was solved using a CHRhelix from the 1+3 complex as a search model. The structure of the 1+10complex was solved using two search models, an NHR helix from the 1+3complex and a CHR helix from the structure of α/β-peptide 10 alone. Thestructure of the 1+8 complex was solved using two search models, an NHRhelix from the 1+3 complex and a chimeric CHR helix prepared from thestructures of 1+3 and 1+10. Molecular graphics were prepared using PyMOL(DeLano Scientific, Palo Alto, Calif., USA).

Protease Stability.

Stock solutions of peptides were prepared at a concentration of 25 μM(based on UV absorbance) in TBS. A solution of proteinase K was preparedat a concentration of 50 μg/mL (based on weight to volume) in TBS. Foreach proteolysis reaction, 40 μL of peptide stock was mixed with 10 μLof proteinase K stock. The reaction was allowed to proceed at roomtemperature and quenched at the desired time point by addition of 100 μLof 1% TFA in water. 125 μL of the resulting quenched reaction wasinjected onto an analytical reverse phase HPLC and run on a gradientbetween 0.1% TFA in water and 0.1% TFA in acetonitrile. The amount ofstarting peptide present quantified by integration of the peak at 220nm. Duplicate reactions were run for each time point. Half-lives weredetermined by fitting time dependent peptide concentration to anexponential decay using GraphPad Prism-brand software (GraphPadSoftware, Inc., La Jolla, Calif., USA). Crude samples for some timepoints were analyzed by MALDI-MS, and the products observed were used toidentify amide bonds cleaved in the course of the reaction.

Expression, Purification, and Refolding of gp41-5.

The sequence of the gp41-5 construct used herein is below.

(SEQ. ID. NO: 24) MSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILSGGSGGWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLGGSGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILSGGSGGWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLGGSGGSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQA RIL

Expression, purification, and refolding of gp41-5 were carried out aspreviously described. Frey, G.; Rits-Volloch, S.; Zhang, X. Q.;Schooley, R. T.; Chen, B.; Harrison, S. C. Proc. Nall. Acad. Sci. U.S.A.2006, 103, 13938-13943. A sample of the gp41-5 plasmid, provided byProf. Stephen Harrison (Harvard University), was transfected into E.coli cells (Rosetta™ strain, Novagen) by electroporation. A singlecolony was grown overnight in 20 mL LB supplemented with 50 μg/mLampicillin (resistance provided by the gp41-5 plasmid) and 30 μg/mLchloramphenicol (resistance provided by the plasmid for rare tRNAsincluded in the Rosetta^(m4) strain). 500 mL of antibiotic supplementedLB was inoculated with 5 mL of the overnight starter culture. Cells weregrown at 37° C. to an OD₆₀₀ of 0.75 and subsequently induced by additionof IPTG to a final concentration of 1 mM. The cells were grown for anadditional 3 hr at 37° C., and then centrifuged at 12,000 g for 15 minat 4° C. The cell pellet was dissolved in ice cold glacial acetic acidand left on ice for 45 min with periodic agitation. The suspension wascentrifuged at 39,000 g for 15 min at 4° C. The supernatant was decantedand lyophilized. The crude protein was purified by preparative HPLC on aC₁₈ column eluted by a gradient between 0.1% TFA in water and 0.1% TFAin acetonitrile. Purified protein was lyophilized and stored at −40° C.until refolding. For refolding, purified gp41-5 (˜2 mg) was dissolved in10 mL of 6 M guanidinium chloride. The resulting solution was dialyzedat room temperature against 0.1 M glyicne pH 3.6 (1×) followed by PBS(2×). Precipitate was removed by centrifugation and the resultingprotein used without further purification.

Fluorescence Polarization.

Fluorescence polarization assays were conducted at room temperature inblack polystyrene plates. All measurements were performed in duplicatewells. The assay buffer was composed of 20 mM phosphate, pH 7.4, 1 mMEDTA, 50 mM NaCl, 0.2 mM NaN₃, 0.5 mg/mL “Pluronic F-68”-brandpolyoxyalkylene ether surfactant. The binding affinity of Flu-C38 forgp41-5 was measured by titrating a fixed concentration of the labeledpeptide (0.2 nM) with increasing concentrations of protein in 384-wellplates with a final volume of 50 μL per well in assay buffer with 1% vvDMSO (added to mimic the conditions of the competition FP experiments).All wells were run in duplicate. The plate was allowed to equilibratefor 30 min and analyzed on an Envision 2100 plate reader. The data werefit using Graphpad Prism software (Graphpad Software Inc., La Jolla,Calif.) to a FP direct-binding model. Roehrl, M. H. A.; Wang, J. Y.;Wagner, G. Biochemistry 2004, 43, 16056-16066. The K_(d) of the tracerwas determined to be 0.4±0.1 nM. The binding affinity measured issomewhat tighter than that previously reported for the gp41-5 Flu-CHRinteraction (K_(d)=3 nM), but the previous study utilized a much higherconcentration of tracer in the direct binding experiment (5 nM). Thelower limit of a K_(d) value that can be accurately determined in adirect binding FP experiment is roughly equal to the concentration oftracer employed. Roehrl, Wang, & Wagner, supra.

Competition fluorescence polarization assays were conducted in black96-well plates. A solution of 2 nM gp41-5, 1 nM Flu-C38 was prepared inFP assay buffer and arrayed into a 96-well plate (100 μLwell). A secondstock plate was prepared containing serial dilutions of peptideinhibitors in DMSO. The peptide stock solutions were transferred to theassay plate (1 μL per well). Each assay plate also included 4 wells eachof the following three controls: (1) 100 μL assay buffer+1 μL DMSO; (2)100 μL of 1 nM Flu-C38+1 μL DMSO as an unbound tracer control; (3) 100μL of the 2 nM protein 1 nM tracer solution +1 μL DMSO as a bound tracercontrol. All experimental conditions were carried out in duplicate, andeach peptide was assayed in 2-3 independent experiments. Data analysiswas carried out in GraphPad Prism. Raw mP data from each experiment werefit to a sigmoidal dose response and normalized to the resultingparameters for the top and bottom of the curve. All experiments includedat least one compound showing complete inhibition at the highestconcentrations tested. Normalized data from multiple independent runs ofthe each oligomer were combined and globally fit to an exact analyticalexpressions for FP competitive binding with K_(i) as the only floatingparameter. The lower bound for K_(i) measurable in the competition FPexperiment was considered to be 0.2 nM, based on the K_(d) of thetracer. See Roehrl, Wang, & Wagner, supra.

Circular Dichroism Spectroscopy.

Circular dichroism measurements were carried out on an Aviv 202SFCircular Dichroism Spectrophotometer. Samples of each peptide wereprepared at 20 μM concentration in PBS. Solutions of 1:1 peptidemixtures were prepared by mixing equal volumes from the same 20 μM stocksolutions used for individual peptide measurements. Spectra wererecorded in a 1 mm cell with a step size of 1 nm and an averaging timeof 5 sec. All spectra are background corrected against buffer measuredin the same cell. Thermal melts were carried out in 5-degree incrementswith an equilibration time of 10 min between each temperature change.Thermal unfolding data were fit to a simple two state folding modelShortie, D.; Meeker, A. K.; Freire, E. Biochemistry 1988, 27, 4761-4768)using GraphPad Prism.

Protease Stability.

Stock solutions of the peptides were prepared at a concentration of 25uM (based on UV absorbance) in TBS. A solution of proteinase K wasprepared at a concentration of 50 μg/mL (based on weight to volume) inTBS. For each proteolysis reaction, 40 μL of peptide stock was mixedwith 10 μL of proteinase K stock. The reaction was allowed to proceed atroom temperature and quenched at the desired time point by addition of100 μL of 1% TFA in water. 125 μL of the resulting quenched reaction wasinjected onto an analytical reverse phase HPLC, and the amount ofstarting peptide present quantified by integration of the peak at 220nm. Duplicate reactions were run for each time point. Half-lives weredetermined by fitting time dependent peptide concentration to anexponential decay using GraphPad Prism. Crude samples for some timepoints were analyzed by MALDI-MS, and the products observed were used toidentify amide bonds cleaved in the course of the reaction.

Antiviral Assays.

A cell-to-cell-fusion assay based on the envelope glycoprotein of theHIV-1 clone HXB2 expressed in CHO cells and with U373-MAGI cells astargets was carried out as previously described. (Deng Y Q, Zheng Q,Ketas T J, Moore J P, & Lu M (2007) Protein design of a bacteriallyexpressed HIV-1 gp41 fusion inhibitor. Biochemistry 46(14):4360-4369.)All the α/β peptides showed no cytotoxicity at 5 μM, as judged bymeasuring the basal level of β-galactosidase expression in the U373-MAGItarget cells. Inhibition of HIV-1 infectivity was measured on TZM-bl(JC53BL) cells, which express CD4, CXCR4, CCR5 and the luciferase geneunder the control of HIV-1 LTR (long terminal repeat). (Wei X P, et al.(2002) Emergence of resistant human immunodeficiency virus type 1 inpatients receiving fusion inhibitor (T-20) monotherapy. AntimicrobAgents Chemother 46(6):1896-1905.) Viral stocks produced in PBMC of fourHIV-1 strains were used: NL4-3, a clone derived from the X4-tropicT-cell line-adapted isolate 111B of Glade B; HC4, an X4 primary isolateof Glade B (Trkola A, et al. (1998) Neutralization sensitivity of humanimmunodeficiency virus type 1 primary isolates to antibodies andCD4-based reagents is independent of coreceptor usage. J Virol72(3):1876-1885); an R5 primary isolate, CC 185 (Glade B) (Connor R I,Sheridan K E, Ceradini D, Choe S, & Landau N R (1997) Change incoreceptor use correlates with disease progression in HIV-1-infectedindividuals. J Exp Med 185 (4): 621-628); and another R⁵ primaryisolate, DJ258 (Glade A) (Louwagie J, et al. (1995) Genetic diversity ofthe envelope glycoprotein from human immunodeficiency virus type-1isolates of African origin. J Virol 69(1):263-271).

Briefly, TZM-bl cells were seeded the day before inoculation at adensity of 10⁵ cellsml, 100 μl/well. Serially diluted peptide in 50 μl(or medium alone as a control) was added to each well. Then the virus,40 TCID₅₀ in 50 μl, or medium only as a background control, was added toeach well. On the third day, the wells were inspected by lightmicroscopy. Wells with and without peptide were compared for cellconfluency and morphology. No signs of toxicity were discerned at thehighest concentrations of peptide used. The infectivity was thenquantified in relative light units with the Bright-Glo Luciferase AssaySystem (Promega Corporation, Madison, Wis., USA), according to themanufacturer's instructions. The experiment was performed three times.The signal of test wells was normalized to that of control wells withoutinhibitor after background subtraction from both. The % inhibition ofinfectivity was expressed as a function of the log₁₀ concentration ofinhibitor in nM. A four-parameter sigmoid function was fitted to thedata in Prism (Graphpad). The R² values for the fits were 0.95-1.0 forNL4-3; 0.98-1.0 for HC4; 0.95-0.98 for CC 185; and 0.92-0.98 for DJ258.Finally, the means±S.E.M. of the IC₅₀ values from the individual fits ofthe three repeat experiments were calculated. The results are depictedgraphically in FIGS. 10A (NL4-3), 10B (CC185), 10C (HC4), and 10D(DJ258).

Sequence-Based Design of α/β-Peptides That Mimic BH3 Domains:

As noted above, designing molecules that bind tightly and selectively toa specific site on a protein constitutes a fundamental challenge inmolecular recognition. Thus, a systematic approach for identifyingsuitable molecules would be a distinct advantage. This Example ispresented to show that systematic backbone modification throughout anatural protein-binding domain (i.e., sequence-based design) can be usedto expeditiously generate α/β-peptide foldamers that bind tightly andselectively to target protein surfaces. In this Example, thesequence-based design approach was used to develop α/β-peptide foldamerligands for the BH3-recognition cleft of the protein Bcl-x_(L).Bcl-x_(L). is a member of the Bcl-2 family, which controls programmedcell death pathways and includes both anti-apoptotic members (e.g.,Bcl-2, Bcl-x_(L), Mcl-1) and pro-apoptotic members (e.g., Bak, Bad,Puma). See Adams & Cory (2007) Oncogene 26:1324-1337.

This Example describes a sequence-based design of α/β-peptide ligandsfor BH3-recognition clefts that differs fundamentally from thestructure-based design approaches to foldamer ligands previously pursuedby the present inventors and others. The approach involves replacingsubsets of regularly spaced α-residues with β-residues bearing theoriginal side chains. Each α to β replacement introduces an extramethylene unit into the backbone. This sequence-based approach does notdirectly aim to recapitulate the folded structure of an α-peptideprototype, although conformational mimicry is achieved as a byproduct ofthe replacement strategy employed. As shown in the earlier Example, ithas been demonstrated that sequence-based design can be used to generatehelix-bundle foldamer quaternary structure from an α-peptide prototype.In this Example, the method is used to mimic the protein-bindingbehavior of an α-helical BH3 domain. The results demonstrate thatsequence-based design is more efficient than structure-based design forgenerating foldamers that bind tightly to the anti-apoptotic Bcl-2family proteins, and that sequence-based design can deliver α/β-peptidesthat display significant resistance to proteolytic degradation.

Puma is a Bcl-2 homolog that binds promiscuously to anti-apoptoticfamily members. See Chen et al. (2005) Mol. Cell. 17:393-403. A26-residue α-peptide corresponding to the Puma BH3 domain (1′) wasprepared, along with seven α/β-peptide analogues (2′-8′) with the sameprimary sequence of side chains displayed on different α/β-peptidebackbones. See FIG. 7A. Each α/β-peptide contained an ααβαααβ backbonerepeat which was derived from the heptad pattern common among α-peptidesequences that form α-helices with a well-developed “stripe” ofhydrophobic side chains running along one side. See FIG. 7B. Recentcrystal structures demonstrate that the ααβαααβ backbone allowsformation of an α-helix-like conformation. See Home, Price, Keck, &Gellman (2007) J. Am. Chem. Soc. 129:4178-4180. α/β-Peptides 2′-8′represent all possible isomers of the Puma BH3 sequence with the ααβαααβbackbone pattern. These oligomers can be viewed as a series of analogsof Puma in which a band of β-residues moves around the helicalperiphery. See FIG. 7C.

Compounds 1′-8′ were tested for their ability to bind to two distinctBcl-2 family targets, Bcl-xL and Mcl-1. Inhibition constants (K_(i) foreach compound were determined by competition fluorescence polarization(FP) assays (see FIG. 8) with a fluorescently labeled Bak-BH3 peptide asthe tracer. The Puma-BH3 peptide (1′) showed affinities for Bcl-xL andMcl-1 that are tighter than can be measured with these FP assays, whichis consistent with previous work. K, values for α/β-peptides 2′-8′ varyfrom less than 1 nM to greater than 100 μM. Variation in the position ofβ-residue incorporation causes considerable changes in affinity for eachprotein: greater than 100,000-fold for Bcl-x_(L) and greater than700-fold for Mcl-1.

For both protein targets, 4′ is the tightest-binding foldamer, with K,<1 nM for Bcl-x_(L) and K_(i)=150 nM for Mcl-1. It is noteworthy thatα/β-peptide 5′, which contains β-modifications at critical hydrophobicresidues in the Puma BH3 sequence, shows nanomolar affinity forBcl-x_(L). These data demonstrate that the location of β-residueincorporation strongly influences Bcl-x_(L) versus Mcl-1 selectivityamong the Puma-derived α/β-peptide isomers, in addition to affinity forthese protein targets. For example, 3′ shows equal affinity for the twoproteins, but 5′ displays greater than 4000-fold selectivity forBcl-x_(L) over Mcl-1. The validity of the conclusions regarding affinityand selectivity derived from the FP competition assays were tested forα-peptide 1′ and α/β-peptides 4′ and 5′ by performing direct-binding FPmeasurements with analogs in which the N-terminal acetyl group isreplaced with a BODIPY-TMR fluorophore. The K_(d) values determined bydirect binding were consistent with the K_(i), values obtained fromcompetition data (see Table 5). The differences in absolute values ofK_(d) versus K_(i) may reflect modest contributions of the appendedfluorophore to affinity as measured in the direct binding mode.

TABLE 5 Binding affinity and protease stability data for α-peptide 1′and α/β-peptides 4′, 5′. K₁[nM]^([a]) K_(d)[nM]^([b]) t_(1/2)[min]^([c])Bcl-x_(L) Mcl-1 Bcl-x_(L) Mcl-1 Prot. K Pronase 1′ <1 <10 <1 <2 0.7 1 2′<1 150 2.2 110 >3000 100 3′ 2.4 11000 1 1100 170 3.5 ^([a])Inhibitionconstants determined by competition FP. ^([b])Dissociation constants ofBODIPY-labeled analogues determined by direct binding FP. ^([c])Measuredhalf-life of a 50 μm solution of α-peptide or α/β-peptide in thepresence of 10 μg mL⁻¹ proteinase K or 5 μg mL⁻¹ pronase.

Having established that certain α/β-analogs of the Puma BH3 domain canbind with high affinity to the natural protein partners, an experimentwas performed to determine whether the α/β-peptides would be recognizedand processed by proteolytic enzymes. α-Peptide 1′ and α/β-peptides 4′and 5′ were tested for their susceptibility to two proteases with broadsubstrate profiles: (1) proteinase K, a non-specific serine proteasethat tends to cleave C-terminal to hydrophobic residues, and (2)“PRONASE”-brand proteinase, a mixture of aggressive endopeptidases andexopeptidases that digests proteins into individual amino acids.(“PRONASE” is a registered trademark of EMD Chemicals, Inc., Gibbstown,N.J.) The results, presented in Table 3, show that the ααβαααβ backbonecan confer substantial resistance to proteolytic degradation.α/β-Peptide 4′, which binds tightly to both Bcl-x_(L) and Mcl-1, showeda greater than 4000-fold improvement in stability to proteinase K and a100-fold improvement in stability to “PRONASE”-brand proteinase relativeto α-peptide 1′. Analysis of the cleavage products by mass spectrometryindicated that the β-residues tend to protect nearby amide groups fromproteolysis, which is consistent with previous reports for isolated a toβ³ insertions. α/β-Peptide 5′ is more susceptible than is isomer 4′ toproteolytic degradation, but 5′ nevertheless shows significantimprovement relative to α-peptide 1′.

Previous work has suggested that the α-helical propensity of BH3-derivedα-peptides may be an important determinant of affinity foranti-apoptotic Bcl-2 family proteins. Circular dichroism (CD)spectroscopy was therefore employed to probe for conformationaldifferences among two of the tight-binding α/β-peptides (4′ and 5′) andone of the weakest binding analogs (7′) described in this Example.Qualitative comparison of CD spectra for 4′, 5′, and 7′ indicates thatthe large differences in binding affinity among these three isomerscannot be explained by differences in helical propensity. Each of thesethree α/β-peptides shows a CD minimum at approximately 202 nm withper-residue ellipticity between −13,000 and −15,000 deg cm² dmol⁻¹ inaqueous solution. Helix formation in the ααβαααβ backbone is reflectedby a strong CD minimum at 206 nm with a maximum magnitude ofapproximately −40,000 deg cm² dmol⁻¹. Thus, the CD data for 4′, 5′, and7′ alone in aqueous solution suggest relatively low population of thehelical state. Similarly, the CD signature for Puma α-peptide 1 inaqueous solution ([θ]₂₂₂=−10,000 deg cm² dmol⁻¹ res⁻¹) suggests littleα-helical content. Without being limited to any specific mechanism, onthe basis of the established precedent for induction of α-helixformation upon binding of BH3 domain α-peptides to Bcl-x_(L) and Mcl-1,the co-inventors hypothesize that α/βpeptides such as 4′ and 5′ areinduced to adopt helical conformations upon binding to protein partners.

The work reported herein demonstrates that a straightforward principleof sequence-based design can be used to convert a helical α-peptideligand into an α/β-peptide with comparable binding affinity for proteintargets and substantially improved proteolytic stability. The strategydisclosed and claimed herein is a fundamental departure from previouswork on the development of foldamer-based inhibitors of protein-proteininteractions. The sequence-based approach disclosed herein has beenshown by these Examples to be more efficient than the structure-basedapproach for generating foldamer mimics of α-helices.

In short, evaluating a series of just seven α/β-peptides designed purelyon the basis of primary sequence information led to a compound thatrivals the best of the previously described chimeric α/β+α ligands inbinding affinity for Bcl-x_(L). See Sadowsky, Schmitt, Lee, Umezawa,Wang, Tomita, and Gellman (2005) J. Am. Chem. Soc 127:11966-11968;Sadowsky, Fairlie, Hadley, Lee, Umezawa, Nikolovska-Coleska, Wang,Huang, Tomita, and Gellman (2007) J. Am. Chem. Soc. 129:139-154; andSadowsky, Murray, Tomita, and Gellman (2007) ChemBioChem 8:903-916.Moreover, the best α/β-peptide binds moderately well to Mcl-1, abiomedically important Bcl-2 family protein that is not targeted byoligomers identified through structure-based design. The implementationof multiple and systematic α-residue to β-residue replacementsthroughout a peptide sequence (7 of 26 positions substituted in the PumaBH3 domain) constitutes a significant advance beyond earlier precedentsin the design of bioactive, proteolytically stable oligomers. Thefinding that one version of this substitution pattern is well-toleratedin terms of binding to anti-apoptotic proteins is surprising andnoteworthy.

The sequence-based design illustrated herein can be implemented withcommercially available α- and β-amino acid monomers and standardautomated peptide synthesis methods. Thus, it is straightforward forothers to undertake analogous efforts.

Comparisons of Chimeric α-α/β Foldamers:

Peptides 12, 13, and 8, below are chimeric α+α/β foldamers of a lead α/βfoldamer 10. These peptides were created to determine the effect of betasubstitution in the region near the N terminus. The beta residues weresequentially subtracted in the“f” and “c” positions along the heptad.The effect of α to β substitutions was monitored with a previouslyreported Fluorescence Polarization (FP) competition assay. (Frey, G.;Rits-Volloch, S.; Zhang, X. Q.; Schooley, R. T.; Chen, B.; Harrison, S.C. Small molecules that bind the inner core of gp41 and inhibit HIVenvelope-mediated fusion. Proc. Natl. Acad. Sci., 2006, 103, 13938-43.)The results suggest that β substitution has a slow, cumulative effect ofdecreasing the binding. Chimeric α+α/β Foldamers, Subtracting β Residuesfrom the “f” and “c” Positions Near the N-Terminus:

   fgabcdefgabcdefg . . .  (SEQ. ID. NO: 10) 10: 

(SEQ. ID. NO: 25) 12: 

(SEQ. ID. NO: 26) 13: 

(SEQ. ID. NO: 8)  8: 

K_(i) (nM): compound 10 = 9 compound 12 = 8 compound 13 = 0.8 compound 8= 0.3

To determine if β substitution disrupted binding in one region of thepeptide, α+α/β chimeric peptides were synthesized with different alphasegments substituted in the beta stripe. The regions of focus were nearthe N terminus 8, middle 14, and C terminus of the peptide 15. The FPdata showed that introducing an alpha segment did increase binding ofthe foldamer; however, the K_(i)'s were all very similar, whichsuggested that β substitution slowly disrupted the binding across theentire length of the helix and not in a particular region.

Chimeric α+α/β Foldamers, Substitution of α Segments in the N-Terminal,Middle, and C-Terminal Regions:

(SEQ.ID. NO: 8)  8: Ac-TTWEAWDRAIAEYA X RIE X LI Z AAQEQQEKNE X AL ZEL-NH₂ (SEQ. ID. NO: 27) 14: Ac-TTWE X WD Z AIAEYAARIEALIRAAQEQQEKNE XAL Z EL-NH₂ (SEQ. ID. NO: 28) 15: Ac-TTWE X WD Z AIAEYA X RIE X LI ZAAQEQQEKNEAALREL-NH₂ K_(i) (nM): compound  8 = 0.3 compound 14 = 1.4compound 15 = 0.2

Foldamer 10 showed that cyclic residues effectively constrained theCα-Cβ torsional angles to aid in folding, but other tactics could beused to constrain a helix. Salt bridges of α residues were effective atpre-forming a helices. See Nishikawa, H.; Nakamura, S.; Kodama, E.; Ito,S.; Kajiwara, K.; Izumi, K.; Sakagami, Y.; Oishi, S.; Ohkubo, T.;Kobayashi, Y.; Otaka, A.; Fujii, N.; Matsuoka, M. Electrostaticallyconstrained alpha-helical peptide inhibits replication of HIV-1resistant to enfuvirtide. Int. J. Biochem. Cell Biol. 2009, 41, 891-9.Another design strategy positioned a stripe of arginines in the iposition which interacted with a stripe of glutamates in the i+4position, favoring an α helical structure. See Burkhard, P.; Meier, M.;Lustig, A. Design of a minimal protein oligomerization domain by astructural approach. Prot. Sci., 2000, 9, 2294-2301.

The following peptide 17 examined the ability of beta residues to formsalt bridges that pre-organize a helix. Because it was previously foundthat the “f” and “c” positions were the most compliant with betasubstitution, β-hArg was placed in the “f” position and β-hGlu wasplaced in the “c” position to maximize i and i+4 interactions. Peptide16 was created to test if both cyclic beta residues and salt bridgingbeta residues worked synergistically in the beta stripe. The FP datasuggested that α/β foldamers 16 and 17 were approximately equalinhibitors to foldamer 10.

(SEQ. ID. NO: 10)        fgabcdefgabcdefg . . . 10: Ac-TTWE X WD ZAIAEYA X RIE X LI Z AAQEQQEKNE X AL Z EL-NH₂ (SEQ. ID. NO: 29)16: Ac-RTWEEWDRAIAEYA X RIE X LI Z AAQ X QQ Z KNE X AL Z EL-NH₂(SEQ. ID. NO: 30) 17: Ac-RTWEEWDRAIAEYARRIEELIRAAQEQQRKNEEALREL-NH₂K_(i) (nM): compound 10 = 9 compound 16 = 3 compound 17 = 11These results are significant in that compound 17 does not contain anycyclically constrained residues. While not being limited to anyunderlying mechanism or phenomenon, it appears that conformationalstability is achieved by incorporating ion pairs along one side of thehelical conformation.

1. A method of fabricating biologically active, unnatural polypeptides,the method comprising: (a) selecting a biologically active polypeptideor biologically active fragment thereof having an amino acid sequencecomprising α-amino acid residues; and (b) fabricating a syntheticpolypeptide that has an amino acid sequence that corresponds to thesequence of the biologically active polypeptide or fragment of step (a),wherein (i) in the synthetic polypeptide between about 14% and about 50%of the α-amino acid residues found in the biologically activepolypeptide or fragment of step (a) are replaced with β-amino acidresidues; (ii) in the synthetic polypeptide the β-amino acid residuesand the α-amino acid residues are distributed in a repeating pattern;and (iii) the synthetic polypeptide has a length of from about 10residues to about 100 residues and comprises at least two β-amino acidresidues.
 2. The method of claim 1, wherein step (b)(i) comprisesreplacing at least one of the α-amino acid residues with at least oneβ-amino acid residue that is cyclically constrained via a ringencompassing its β² and β³ carbon atoms.
 3. The method of claim 1,wherein step (b)(i) comprises replacing between about 14% and about 50%of the α-amino acid residues found in the biologically activepolypeptide or fragment of step (a) with β-amino acid residues that arecyclically constrained via a ring encompassing their β² and β³ carbonatoms.
 4. The method of claim 1, wherein step (b)(i) comprises replacingbetween about 14% and about 50% of the α-amino acid residues found inthe biologically active polypeptide or fragment of step (a) with β-aminoacid residues, wherein at least one of the 13-amino acid residues isunsubstituted at its β² and β³ carbon atoms.
 5. The method of claim 1,wherein step (b)(i) comprises replacing between about 14% and about 50%of the α-amino acid residues found in the biologically activepolypeptide or fragment of step (a) with β-amino acid residues whereinall of the β-amino acid residues are substituted at their β² and β³carbon atoms.
 6. The method of claim 1, wherein step (b)(i) comprisesreplacing between about 14% and about 50% of the α-amino acid residuesfound in the biologically active polypeptide or fragment of step (a)with β-amino acid residues wherein each β-amino acid residue has atleast one side chain identical to the α-amino acid residue it replaces.7. A method of fabricating biologically active, proteoloytic-resistant,unnatural polypeptides, the method comprising: (a) selecting abiologically active polypeptide or biologically active fragment thereofhaving an amino acid sequence comprising α-amino acid residues; and (b)fabricating a synthetic polypeptide that has an amino acid sequence thatcorresponds to the sequence of the biologically active polypeptide orfragment of step (a), wherein (i) in the synthetic polypeptide betweenabout 14% and about 50% of the α-amino acid residues found in thebiologically active polypeptide or fragment of step (a) are replacedwith analogous β-amino acid residues; (ii) each analogous β-amino acidresidue has at least one side chain identical to the α-amino acidresidue it replaces; (iii) in the synthetic polypeptide the β-amino acidresidues and the α-amino acid residues are distributed in a repeatingpattern and (iv) the synthetic polypeptide has a length of from about 10residues to about 100 residues and comprises at least two β-amino acidresidues.
 8. The method of fabricating biologically active, unnaturalpolypeptides according to any one of claims 1 to 7, wherein in a foldedstructure adopted by the polypeptides, the repeating pattern disposesthe β-amino acid residues in alignment along one side of the foldedmolecular structure when the unnatural polypeptides adopt a helicalconformation.
 9. The method of fabricating biologically active,unnatural polypeptides according to any one of claims 1 to 7, whereinthe repeating pattern of β-amino acid residues and α-amino acid residuesis selected from the group consisting of (ααααααβ), (αααααβ), (ααααβ),(αααβ), (ααβ), (ααβαααβ), (ααβαβαβ), and (α/β).
 10. The method offabricating biologically active, unnatural polypeptides according to anyone of claims 1 to 7, wherein step (b) comprises fabricating a syntheticpolypeptide having between about 20 residues and about 50 residues. 11.An isolated, unnatural polypeptide comprising a primary amino acidsequence as shown in SEQ. ID. NOS: 4-11, 16-22, and 25-30.
 12. A methodof inhibiting fusion of human immunodeficiency virus to human cells, themethod comprising contacting human cells with an isolated, unnaturalpolypeptide comprising a primary amino acid sequence as shown in SEQ.ID. NOS: 4-11 and 25-30.
 13. A method of inhibiting fusion of humanimmunodeficiency virus (HIV) to human cells, the method comprising: (a)selecting a natural, biologically active polypeptide or biologicallyactive fragment thereof having an amino acid sequence comprising α-aminoacid residues, and necessary for HIV fusion in vivo; (b) fabricating asynthetic polypeptide that has an amino acid sequence that correspondsto the sequence of the biologically active polypeptide or fragment ofstep (a), wherein (i) in the synthetic polypeptide between about 14% andabout 50% of the α-amino acid residues found in the biologically activepolypeptide or fragment of step (a) are replaced with β-amino acidresidues; (ii) in the synthetic polypeptide the β-amino acid residuesand the α-amino acid residues are distributed in a repeating pattern;and (iii) the synthetic polypeptide has a length of from about 10residues to about 100 residues, and comprises at least two β-amino acidresidues; and then (c) contacting human cells with the syntheticpolypeptide of step (b).
 14. A pharmaceutical composition comprising:(i) a biologically active polypeptide or pharmaceutical salt thereof;and (ii) a pharmaceutically acceptable carrier or diluent, wherein thebiologically active polypeptide or pharmaceutical salt thereof comprisesa repeated pattern of one or more α-amino acid residues and one or moreβ-amino acid residues.
 15. The pharmaceutical composition of claim 14,wherein the biologically active polypeptide or pharmaceutical saltthereof comprises between about 7% to about 50% β-amino acid residues;and wherein the biologically active polypeptide or pharmaceutical saltthereof comprises a repeated pattern of one or more α-amino acidresidues and one or more β-amino acid residues.
 16. The pharmaceuticalcomposition of claim 14, wherein the repeated pattern of β-amino acidresidues and α-amino acid residues is selected from the group consistingof (ααααααβ), (αααααβ), (ααααβ), (αααβ), (βαβ), (ααβαααβ), (ααβαβαβ),and (αβ).
 17. The pharmaceutical composition of claim 14, wherein thebiologically active polypeptide or pharmaceutical salt thereof comprisesbetween about 10 residues to about 100 residues and comprises at leasttwo β-amino acid residues.
 18. The pharmaceutical composition of claim14, wherein the biologically active polypeptide or pharmaceutical saltthereof comprises at least one β-amino acid residue that is cyclicallyconstrained via a ring encompassing its β² and β³ carbon atoms.
 19. Thepharmaceutical composition of claim 14, wherein at least one of theβ-amino acid residues is unsubstituted at its β² or β³ carbon atoms. 20.The pharmaceutical composition of claim 14, wherein each β-amino acidresidue has at least one side chain identical to the α-amino acidresidue it replaces.
 21. The pharmaceutical composition of claim 14,wherein, in a folded structure adopted by the polypeptide, the repeatingpattern disposes the β-amino acid residues in alignment along one sideof the folded molecular structure when the unnatural polypeptides adopta helical conformation.
 22. The pharmaceutical composition of claim 14,wherein the biologically active polypeptide or pharmaceutical saltthereof comprises between about 20 residues and about 50 residues. 23.The pharmaceutical composition of claim 14, wherein the biologicallyactive polypeptide or pharmaceutical salt thereof comprises an aminoacid sequence selected from SEQ. ID. NOS: 4-11, 16-22, and 25-30. 24.The pharmaceutical composition of claim 14, wherein the biologicallyactive polypeptide or pharmaceutical salt thereof comprises SEQ. ID. NO:10.